Unimolecular segment amplification and sequencing

ABSTRACT

Disclosed are compositions and a method for amplification of and multiplex detection of molecules of interest involving rolling circle replication. The method is useful for simultaneously detecting multiple specific nucleic acids in a sample with high specificity and sensitivity. The method also has an inherently low level of background signal. A preferred form of the method consists of an association operation, an amplification operation, and a detection operation. The association operation involves association of one or more specially designed probe molecules, either wholly or partly nucleic acid, to target molecules of interest. This operation associates the probe molecules to a target molecules present in a sample. The amplification operation is rolling circle replication of circular nucleic acid molecules, termed amplification target circles, that are either a part of, or hybridized to, the probe molecules. A single round of amplification using rolling circle replication results in a large amplification of the amplification target circles. Following rolling circle replication, the amplified sequences are detected using combinatorial multicolor coding probes that allow separate, simultaneous, and quantitative detection of multiple different amplified target circles representing multiple different target molecules. Since the amplified product is directly proportional to the amount of target sequence present in a sample, quantitative measurements reliably represent the amount of a target sequence in a sample. Major advantages of this method are that a large number of distinct target molecules can be detected simultaneously, and that differences in the amounts of the various target molecules in a sample can be accurately quantified. It is also advantageous that the DNA replication step is isothermal, and that signals are strictly quantitative because the amplification reaction is linear and is catalyzed by a highly processive enzyme.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Application Ser. No.08/563,912, filed Nov. 21, 1995 now U.S. Pat. No. 5,854,033. Thisapplication also claims benefit of U.S. Provisional Application No.60/016,677, filed May 1, 1996.

BACKGROUND OF THE INVENTION

The disclosed invention is generally in the field of assays fordetection of nucleic acids, and specifically in the field of nucleicacid amplification and sequencing.

A number of methods have been developed which permit the implementationof extremely sensitive diagnostic assays based on nucleic aciddetection. Most of these methods employ exponential amplification oftargets or probes. These include the polymerase chain reaction (PCR),ligase chain reaction (LCR), self-sustained sequence replication (3SR),nucleic acid sequence based amplification (NASBA), strand displacementamplification (SDA), and amplification with Qβ replicase (Birkenmeyerand Mushahwar, J. Virological Methods, 35:117-126 (1991); Landegren,Trends Genetics, 9:199-202 (1993)).

While all of these methods offer good sensitivity, with a practicallimit of detection of about 100 target molecules, all of them sufferfrom relatively low precision in quantitative measurements. This lack ofprecision manifests itself most dramatically when the diagnostic assayis implemented in multiplex format, that is, in a format designed forthe simultaneous detection of several different target sequences.

In practical diagnostic applications it is desirable to assay for manytargets simultaneously. Such multiplex assays are typically used todetect five or more targets. It is also desirable to obtain accuratequantitative data for the targets in these assays. For example, it hasbeen demonstrated that viremia can be correlated with disease status forviruses such as HIV-1 and hepatitis C (Lefrere et al., Br. J. Haematol.,82(2):467-471 (1992), Gunji et al., Int. J. Cancer, 52(5):726-730(1992), Hagiwara et al., Hepatology, 17(4):545-550 (1993), Lu et al., J.Infect. Dis., 168(5):1165-8116 (1993), Piatak et al., Science,259(5102):1749-1754 (1993), Gupta et al., Ninth International Conferenceon AIDS/Fourth STD World Congress, Jun. 7-11, 1993, Berlin, Germany,Saksela et al., Proc. Natl. Acad. Sci. USA, 91(3):1104-1108 (1994)). Amethod for accurately quantitating viral load would be useful.

In a multiplex assay, it is especially desirable that quantitativemeasurements of different targets accurately reflect the true ratio ofthe target sequences. However, the data obtained using multiplexed,exponential nucleic acid amplification methods is at bestsemi-quantitative. A number of factors are involved:

1. When a multiplex assay involves different priming events fordifferent target sequences, the relative efficiency of these events mayvary for different targets. This is due to the stability and structuraldifferences between the various primers used.

2. If the rates of product strand renaturation differ for differenttargets, the extent of competition with priming events will not be thesame for all targets.

3. For reactions involving multiple ligation events, such as LCR, theremay be small but significant differences in the relative efficiency ofligation events for each target sequence. Since the ligation events arerepeated many times, this effect is magnified.

4. For reactions involving reverse transcription (3SR, NASBA) or klenowstrand displacement (SDA), the extent of polymerization processivity maydiffer among different target sequences.

5. For assays involving different replicatable RNA probes, thereplication efficiency of each probe is usually not the same, and hencethe probes compete unequally in replication reactions catalyzed by Qβreplicase.

6. A relatively small difference in yield in one cycle of amplificationresults in a large difference in amplification yield after severalcycles. For example, in a PCR reaction with 25 amplification cycles anda 10% difference in yield per cycle, that is, 2-fold versus 1.8-foldamplification per cycle, the yield would be 2.0²⁵ =33,554,000 versus1.8²⁵ =2,408,800. The difference in overall yield after 25 cycles is14-fold. After 30 cycles of amplification, the yield difference would bemore than 20-fold.

Accordingly, there is a need for amplification methods that are lesslikely to produce variable and possibly erroneous signal yields inmultiplex assays.

It is therefore an object of the disclosed invention to provide a methodof amplifying diagnostic nucleic acids with amplification yieldsproportional to the amount of a target sequence in a sample.

It is another object of the disclosed invention to provide a method ofdetecting specific target nucleic acid sequences present in a samplewhere detection efficiency is not dependent on the structure of thetarget sequences.

It is another object of the disclosed invention to provide a method ofdetermining the amount of specific target nucleic acid sequences presentin a sample where the signal level measured is proportional to theamount of a target sequence in a sample and where the ratio of signallevels measured for different target sequences substantially matches theratio of the amount of the different target sequences present in thesample.

It is another object of the disclosed invention to provide a method ofdetecting and determining the amount of multiple specific target nucleicacid sequences in a single sample where the ratio of signal levelsmeasured for different target nucleic acid sequences substantiallymatches the ratio of the amount of the different target nucleic acidsequences present in the sample.

It is another object of the disclosed invention to provide a method ofdetecting the presence of single copies of target nucleic acid sequencesin situ.

It is another object of the disclosed invention to provide a method ofdetecting the presence of target nucleic acid sequences representingindividual alleles of a target genetic element.

It is another object of the disclosed invention to provide a method fordetecting, and determining the relative amounts of, multiple moleculesof interest in a sample.

It is another object of the disclosed invention to provide a method fordetermining the sequence of a target nucleic acid sequence.

It is another object of the present invention to provide a method ofdetermining the range of sequences present in a mixture of targetnucleic acid sequences.

SUMMARY OF THE INVENTION

Disclosed are compositions and a method for amplifying nucleic acidsequences based on the presence of a specific target sequence oranalyte. The method is useful for detecting specific nucleic acids oranalytes in a sample with high specificity and sensitivity. The methodalso has an inherently low level of background signal. Preferredembodiments of the method consist of a DNA ligation operation, anamplification operation, and, optionally, a detection operation. The DNAligation operation circularizes a specially designed nucleic acid probemolecule. This step is dependent on hybridization of the probe to atarget sequence and forms circular probe molecules in proportion to theamount of target sequence present in a sample. The amplificationoperation is rolling circle replication of the circularized probe. Asingle round of amplification using rolling circle replication resultsin a large amplification of the circularized probe sequences, orders ofmagnitude greater than a single cycle of PCR replication and otheramplification techniques in which each cycle is limited to a doubling ofthe number of copies of a target sequence. Rolling circle amplificationcan also be performed independently of a ligation operation. By couplinga nucleic acid tag to a specific binding molecule, such as an antibody,amplification of the nucleic acid tag can be used to detect analytes ina sample. This is preferred for detection of analytes where anamplification target circle serves as an amplifiable tag on a reporterbinding molecule, or where an amplification target circle is amplifiedusing a rolling circle replication primer that is part of a reporterbinding molecule. Optionally, an additional amplification operation canbe performed on the DNA produced by rolling circle replication.

Following amplification, the amplified sequences can be detected andquantified using any of the conventional detection systems for nucleicacids such as detection of fluorescent labels, enzyme-linked detectionsystems, antibody-mediated label detection, and detection of radioactivelabels. Since the amplified product is directly proportional to theamount of target sequence present in a sample, quantitative measurementsreliably represent the amount of a target sequence in a sample. Majoradvantages of this method are that the ligation operation can bemanipulated to obtain allelic discrimination, the amplificationoperation is isothermal, and signals are strictly quantitative becausethe amplification reaction is linear and is catalyzed by a highlyprocessive enzyme. In multiplex assays, the primer oligonucleotide usedfor DNA replication can be the same for all probes.

Following amplification, the nucleotide sequence of the amplifiedsequences can be determined either by conventional means or by primerextension sequencing of amplified target sequence. Two preferred modesof primer extension sequencing are disclosed. Unimolecular SegmentAmplification and Sequencing (USA-SEQ), a form of single nucleotideprimer extension sequencing, involves interrogation of a singlenucleotide in an amplified target sequence by incorporation of aspecific and identifiable nucleotide based on the identity of theinterrogated nucleotide. Unimolecular Segment Amplification and CAGESequencing (USA-CAGESEQ), a form of degenerate probe primer extensionsequencing, involves sequential addition of degenerate probes to aninterrogation primer hybridized to amplified target sequences. Additionof multiple probes is prevented by the presence of a removable cage atthe 3' end. After addition of the degenerate probes, the cage is removedand further degenerate probes can be added or, as the final operation,the nucleotide next to the end of the interrogation primer or the lastadded degenerate probe is interrogated as in USA-SEQ to determine itsidentity. The disclosed primer extension sequencing methods are usefulfor identifying the presence of multiple distinct sequences in a mixtureof target sequences.

The disclosed method has two features that provide simple, quantitative,and consistent amplification and detection of a target nucleic acidsequence. First, target sequences are amplified via a small diagnosticprobe with an arbitrary primer binding sequence. This allows consistencyin the priming and replication reactions, even between probes havingvery different target sequences. Second, amplification takes place notin cycles, but in a continuous, isothermal replication: rolling circlereplication. This makes amplification less complicated and much moreconsistent in output.

Also disclosed are compositions and a method for of multiplex detectionof molecules of interest involving rolling circle replication. Themethod is useful for simultaneously detecting multiple specific nucleicacids in a sample with high specificity and sensitivity. The method alsohas an inherently low level of background signal. A preferred form ofthe method consists of an association operation, an amplificationoperation, and a detection operation. The association operation involvesassociation of one or more specially designed probe molecules, eitherwholly or partly nucleic acid, to target molecules of interest. Thisoperation associates the probe molecules to a target molecules presentin a sample. The amplification operation is rolling circle replicationof circular nucleic acid molecules, termed amplification target circles,that are either a part of, or hybridized to, the probe molecules. Asingle round of amplification using rolling circle replication resultsin a large amplification of the amplification target circles, orders ofmagnitude greater than a single cycle of PCR replication and otheramplification techniques in which each cycle is limited to a doubling ofthe number of copies of a target sequence. By coupling a nucleic acidtag to a specific binding molecule, such as an antibody, amplificationof the nucleic acid tag can be used to detect analytes in a sample.

Following rolling circle replication, the amplified sequences can bedetected using combinatorial multicolor coding probes that allowseparate, simultaneous, and quantitative detection of multiple differentamplified target sequences representing multiple different targetmolecules. Since the amplified product is directly proportional to theamount of target sequence present in a sample, quantitative measurementsreliably represent the amount of a target sequence in a sample. Majoradvantages of this method are that a large number of distinct targetmolecules can be detected simultaneously, and that differences in theamounts of the various target molecules in a sample can be accuratelyquantified. It is also advantageous that the DNA replication step isisothermal, and that signals are strictly quantitative because theamplification reaction is linear and is catalyzed by a highly processiveenzyme.

The disclosed method has two features that provide simple, quantitative,and consistent detection of multiple target molecules. First,amplification takes place not in cycles, but in a continuous, isothermalreplication: rolling circle replication. This makes amplification lesscomplicated and much more consistent in output. Second, combinatorialmulticolor coding allows sensitive simultaneous detection of a largenumber different target molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of an example of an open circle probe hybridized toa target sequence. The diagram shows the relationship between the targetsequence and the right and left target probes.

FIG. 2 is a diagram of an example of a gap oligonucleotide and an opencircle probe hybridized to a target sequence. The diagram shows therelationship between the target sequence, the gap oligonucleotide, andthe right and left target probes.

FIG. 3 is a diagram of an open circle probe hybridized and ligated to atarget sequence. The diagram shows how the open circle probe becomestopologically locked to the target sequence.

FIG. 4 is a diagram of rolling circle amplification of an open circleprobe topologically locked to the nucleic acid containing the targetsequence.

FIG. 5 is a diagram of an example of an open circle probe. Variousportions of the open circle probe are indicated by different fills.

FIG. 6 is a diagram of tandem sequence DNA (TS-DNA) and an address probedesigned to hybridize to the portion of the TS-DNA corresponding to partof the right and left target probes of the open circle probe and the gapoligonucleotide. The TS-DNA is SEQ ID NO:2 and the address probe is SEQID NO:3.

FIG. 7 is a diagram of the capture and detection of TS-DNA. Capture iseffected by hybridization of the TS-DNA to address probes attached to asolid-state detector. Detection is effected by hybridization ofsecondary detection probes to the captured TS-DNA. Portions of theTS-DNA corresponding to various portions of the open circle probe areindicated by different fills.

FIG. 8 is a diagram of an example of ligation-mediated rolling circlereplication followed by transcription (LM-RCT). Diagramed at the top isa gap oligonucleotide and an open circle probe, having a primercomplement portion and a promoter portion next to the right and lefttarget probe portions, respectively, hybridized to a target sequence.Diagramed at the bottom is the rolling circle replication producthybridized to unligated copies of the open circle probe and gapoligonucleotide. This hybridization forms the double-stranded substratefor transcription.

FIG. 9 is a diagram of an example of a multiplex antibody assayemploying open circle probes and LM-RCT for generation of an amplifiedsignal. Diagramed are three reporter antibodies, each with a differentoligonucleotide as a DNA tag. Diagramed at the bottom is amplificationof only that DNA tag coupled to a reporter antibody that bound.

FIG. 10 is a diagram of two schemes for multiplex detection of specificamplified nucleic acids. Diagramed at the top is hybridization ofdetection probes with different labels to amplified nucleic acids.Diagramed at the bottom is hybridization of amplified nucleic acid to asolid-state detector with address probes for the different possibleamplification products attached in a pattern.

FIGS. 11A and 11B are diagrams of an example of secondary DNA stranddisplacement. Diagramed at the top of FIG. 11A is a gap oligonucleotideand an open circle probe hybridized to a target sequence. Diagramed atthe bottom of FIG. 11A is the rolling circle replication producthybridized to secondary DNA strand displacement primers. Diagramed inFIG. 11B is secondary DNA strand displacement initiated from multipleprimers. FIG. 11B illustrates secondary DNA strand displacement carriedout simultaneously with rolling circle replication.

FIG. 12 is a diagram of an example of nested RCA using an unamplifiedfirst open circle probe as the target sequence. Diagramed at the top isa gap oligonucleotide and a first open circle probe hybridized to atarget sequence, and a secondary open circle probe hybridized to thefirst open circle probe. Diagramed at the bottom is the rolling circlereplication product of the secondary open circle probe.

FIG. 13 is a diagram of an example of strand displacement cascadeamplification. Diagramed is the synthesis and template relationships offour generations of TS-DNA. TS-DNA-1 is generated by rolling circlereplication primed by the rolling circle replication primer. TS-DNA-2and TS-DNA-4 are generated by secondary DNA strand displacement primedby a secondary DNA strand displacement primer (P2). TS-DNA-3 isgenerated by strand-displacing secondary DNA strand displacement primedby a tertiary DNA strand displacement primer (P1).

FIG. 14 is a diagram of an example of opposite strand amplification.Diagramed are five different stages of the reaction as DNA synthesisproceeds. TS-DNA-2 is generated by secondary DNA strand displacement ofTS-DNA primed by the secondary DNA strand displacement primer. Asrolling circle replication creates new TS-DNA sequence, the secondaryDNA strand displacement primer hybridizes to the newly synthesized DNAand primes synthesis of another copy of TS-DNA-2.

FIG. 15 is a diagram of an open circle probe including a gap sequence.The lower half of the diagram illustrates a preferred relationshipbetween sequences in the open circle probe and interrogation primers.

FIGS. 16A, 16B, and 16C are diagrams showing the results of unimolecularsegment amplification and sequencing (USA-SEQ) performed on threedifferent nucleic acid samples. The large circles represent a targetsample dot on a solid-state support. The small circles representindividual TS-DNA molecules, amplified in situ at the location of targetnucleic acids in the sample, which have been subjected to primerextension sequencing. FIG. 16A is representative of a sample that ishomozygous for the wild type sequence (indicated by incorporation ofcystine). FIG. 16B is representative of a sample that is heterozygousfor the wild type and a mutant (indicated by an equal number of TS-DNAmolecules resulting in incorporation of cystine and adenine). FIG. 16Cis representative of a sample that is homozygous but includes a fewcells with a somatic mutation.

FIGS. 17A and 17B are diagrams of an example of the relationship of anopen circle probe to two target sequences having a different amount of arepeating sequence. The hybridization of the left target probe and theright target probe of the open circle probe to the two different targetsequences is shown (with | indicating hydrogen bonding). The fillsequences are the nucleotides, complementary to the sequence in thetarget sequence opposite the gap space, which will fill the gap spacebetween the left and right target probes to join the open circle probeinto an amplification target circle. The sequences depicted in thediagrams relate to the assay described in Example 10. In FIG. 17A, thetarget sequence is SEQ ID NO:24, the left target sequence is nucleotides76 to 96 of SEQ ID NO:25, the right target sequence is nucleotides 1 to24 of SEQ ID NO:25, and the fill sequence is nucleotides 97 to 128 ofSEQ ID NO:25. In FIG. 17B, the target sequence is SEQ ID NO:23, the lefttarget sequence is nucleotides 2 to 21 of SEQ ID NO:18, the right targetsequence is nucleotides 1 to 24 of SEQ ID NO:25, and the fill sequenceis nucleotides 22 to 51 of SEQ ID NO:18.

FIGS. 18A, 18B, 18C, 18D, and 18E are diagrams showing a slidecontaining an array of nucleic acid samples and coverage of rows ofsamples with a mask during unimolecular segment amplification and cagesequencing (USA-CAGESEQ).

FIG. 19 is a diagram showing the nucleotide incorporated in the firstcolumn of samples on a slide subjected to USA-CAGESEQ. The samplescorrespond to the target sequence shown in FIG. 17A.

FIG. 20 is a diagram showing the nucleotide incorporated in the firstcolumn of samples on a slide subjected to USA-CAGESEQ. The samplescorrespond to the target sequence shown in FIG. 17B.

FIGS. 21A, 21B, 21C, 21D, 21E, 22A, 22B, 22C, 22D, 22E, 23A, 23B, 23C,23D, 23E, 24A, 24B, 24C, 24D, and 24E depict interrogation primers,formed from interrogation probes and degenerate probes, hybridized toTS-DNA. The figures depict five slides and TS-DNA representing a singlecolumn of five sample dots from each slide. In each row, the top(shorter) sequence is the interrogation primer and the bottom (longer)sequence is a portion of the TS-DNA. The non-underlined portions of theinterrogation primers represent the interrogation probe. The underlinedportions of the interrogation primers were formed by sequential ligationof one or more degenerate probes to the end of the interrogation probe.The nucleotide in boldface is the nucleotide added to the interrogationprimer during primer extension. The TS-DNA sequences shown in FIGS. 21A,21B, 21C, 21D, 21E, 22A, 22B, 22C, 22D, and 22E are related to thetarget sequence shown in FIG. 17A and correspond to nucleotides 1 to 60of SEQ ID NO:19. The interrogation primer sequences in FIGS. 21A, 21B,21C, 21D, 21E, 22A, 22B, 22C, 22D, and 22E correspond to variousportions of nucleotides 76 to 125 of SEQ ID NO:25. The sequences shownin FIGS. 23A, 23B, 23C, 23D, 23E, 24A, 24B, 24C, 24D, and 24E arerelated to the target sequence shown in FIG. 17B and correspond tonucleotides 1 to 58 of SEQ ID NO:26. The interrogation primer sequencesin FIGS. 23A, 23B, 23C, 23D, 23E, 24A, 24B, 24C, 24D, and 24E correspondto various portions of nucleotides 1 to 50 of SEQ ID NO:18.

FIGS. 25A and 25B are diagrams of a reporter binding molecule made up ofa peptide nucleic acid (as the affinity portion) and a rolling circlereplication primer (as the oligonucleotide portion). The affinityportion is shown hybridized to a target DNA. In FIG. 25B, anamplification target circle is shown hybridized to the oligonucleotideportion (that is, the rolling circle replication primer).

FIGS. 26A and 26B are diagrams of a reporter binding molecule hybridizedto a ligated open circle probe that is topologically locked to targetDNA. The reporter binding molecule made up of a peptide nucleic acid (asthe affinity portion) and a rolling circle replication primer (as theoligonucleotide portion). In FIG. 26B, an amplification target circle isshown hybridized to the oligonucleotide portion (that is, the rollingcircle replication primer).

FIGS. 27A and 27B are diagrams of a reporter binding molecule made up ofa chemically-linked triple helix-forming oligonucleotide (as theaffinity portion) and a rolling circle replication primer as theoligonucleotide portion. The affinity portion is shown hybridized to atarget DNA. PS indicates a psoralen derivative creating a chemical linkbetween the affinity portion and the target DNA. In FIG. 27B, anamplification target circle is shown hybridized to the oligonucleotideportion (that is, the rolling circle replication primer).

FIGS. 28A and 28B are diagrams of a reporter binding molecule hybridizedto a ligated open circle probe that is topologically locked to targetDNA. The reporter binding molecule made up of a chemically-linked triplehelix-forming oligonucleotide (as the affinity portion) and a rollingcircle replication primer as the oligonucleotide portion. PS indicates apsoralen derivative creating a chemical link between the affinityportion and the target DNA. In FIG. 28B, an amplification target circleis shown hybridized to the oligonucleotide portion (that is, the rollingcircle replication primer).

FIGS. 29A and 29B are diagrams of a reporter binding molecule made up ofan antibody (as the affinity portion) and a rolling circle replicationprimer (as the oligonucleotide portion). The affinity portion is shownbound to a target antigen. In FIG. 29B, an amplification target circleis shown hybridized to the oligonucleotide portion (that is, the rollingcircle replication primer).

DETAILED DESCRIPTION OF THE INVENTION

The disclosed composition and method make use of certain materials andprocedures which allow consistent and quantitative amplification anddetection of target nucleic acid sequences. These materials andprocedures are described in detail below.

Some major features of the disclosed method are:

1. The ligation operation can be manipulated to obtain allelicdiscrimination, especially with the use of a gap-filling step.

2. The amplification operation is isothermal.

3. Signals can be strictly quantitative because in certain embodimentsof the amplification operation amplification is linear and is catalyzedby a highly processive enzyme. In multiplex assays, the primer used forDNA replication is the same for all probes.

4. Modified nucleotides or other moieties may be incorporated during DNAreplication or transcription.

5. The amplification product is a repetitive DNA molecule, and maycontain arbitrarily chosen tag sequences that are useful for detection.

I. MATERIALS

A. Open Circle Probes

An open circle probe (OCP) is a linear single-stranded DNA molecule,generally containing between 50 to 1000 nucleotides, preferably betweenabout 60 to 150 nucleotides, and most preferably between about 70 to 100nucleotides. The OCP has a 5' phosphate group and a 3' hydroxyl group.This allows the ends to be ligated using a DNA ligase, or extended in agap-filling operation. Portions of the OCP have specific functionsmaking the OCP useful for RCA and LM-RCA. These portions are referred toas the target probe portions, the primer complement portion, the spacerregion, the detection tag portions, the secondary target sequenceportions, the address tag portions, and the promoter portion (FIG. 5).The target probe portions and the primer complement portion are requiredelements of an open circle probe. The primer complement portion is partof the spacer region. Detection tag portions, secondary target sequenceportions, and promoter portions are optional and, when present, are partof the spacer region. Address tag portions are optional and, whenpresent, may be part of the spacer region. Generally, an open circleprobe is a single-stranded, linear DNA molecule comprising, from 5' endto 3' end, a 5' phosphate group, a right target probe portion, a spacerregion, a left target probe portion, and a 3' hydroxyl group, with aprimer complement portion present as part of the spacer region. Thosesegments of the spacer region that do not correspond to a specificportion of the OCP can be arbitrarily chosen sequences. It is preferredthat OCPs do not have any sequences that are self-complementary. It isconsidered that this condition is met if there are no complementaryregions greater than six nucleotides long without a mismatch or gap. Itis also preferred that OCPs containing a promoter portion do not haveany sequences that resemble a transcription terminator, such as a run ofeight or more thymidine nucleotides.

The open circle probe, when ligated and replicated, gives rise to a longDNA molecule containing multiple repeats of sequences complementary tothe open circle probe. This long DNA molecule is referred to herein astandem sequences DNA (TS-DNA). TS-DNA contains sequences complementaryto the target probe portions, the primer complement portion, the spacerregion, and, if present on the open circle probe, the detection tagportions, the secondary target sequence portions, the address tagportions, and the promoter portion. These sequences in the TS-DNA arereferred to as target sequences (which match the original targetsequence), primer sequences (which match the sequence of the rollingcircle replication primer), spacer sequences (complementary to thespacer region), detection tags, secondary target sequences, addresstags, and promoter sequences.

A particularly preferred embodiment is an open circle probe of 70 to 100nucleotides including a left target probe of 20 nucleotides and a righttarget probe of 20 nucleotides. The left target probe and right targetprobe hybridize to a target sequence leaving a gap of five nucleotides,which is filled by a single pentanucleotide gap oligonucleotide.

1. Target Probe Portions

There are two target probe portions on each OCP, one at each end of theOCP. The target probe portions can each be any length that supportsspecific and stable hybridization between the target probes and thetarget sequence. For this purpose, a length of 10 to 35 nucleotides foreach target probe portion is preferred, with target probe portions 15 to20 nucleotides long being most preferred. The target probe portion atthe 3' end of the OCP is referred to as the left target probe, and thetarget probe portion at the 5' end of the OCP is referred to as theright target probe. These target probe portions are also referred toherein as left and right target probes or left and right probes. Thetarget probe portions are complementary to a target nucleic acidsequence.

The target probe portions are complementary to the target sequence, suchthat upon hybridization the 5' end of the right target probe portion andthe 3' end of the left target probe portion are base-paired to adjacentnucleotides in the target sequence, with the objective that they serveas a substrate for ligation (FIG. 1). Optionally, the 5' end and the 3'end of the target probe portions may hybridize in such a way that theyare separated by a gap space. In this case the 5' end and the 3' end ofthe OCP may only be ligated if one or more additional oligonucleotides,referred to as gap oligonucleotides, are used, or if the gap space isfilled during the ligation operation. The gap oligonucleotides hybridizeto the target sequence in the gap space to a form continuousprobe/target hybrid (FIG. 2). The gap space may be any length desiredbut is generally ten nucleotides or less. It is preferred that the gapspace is between about three to ten nucleotides in length, with a gapspace of four to eight nucleotides in length being most preferred.Alternatively, a gap space could be filled using a DNA polymerase duringthe ligation operation (see Example 3). When using such a gap-fillingoperation, a gap space of three to five nucleotides in length is mostpreferred. As another alternative, the gap space can be partiallybridged by one or more gap oligonucleotides, with the remainder of thegap filled using DNA polymerase.

2. Primer Complement Portion

The primer complement portion is part of the spacer region of an opencircle probe. The primer complement portion is complementary to therolling circle replication primer (RCRP). Each OCP should have a singleprimer complement portion. This allows rolling circle replication toinitiate at a single site on ligated OCPs. The primer complement portionand the cognate primer can have any desired sequence so long as they arecomplementary to each other. In general, the sequence of the primercomplement can be chosen such that it is not significantly similar toany other portion of the OCP. The primer complement portion can be anylength that supports specific and stable hybridization between theprimer complement portion and the primer. For this purpose, a length of10 to 35 nucleotides is preferred, with a primer complement portion 16to 20 nucleotides long being most preferred. The primer complementportion can be located anywhere within the spacer region of an OCP. Itis preferred that the primer complement portion is adjacent to the righttarget probe, with the right target probe portion and the primercomplement portion preferably separated by three to ten nucleotides, andmost preferably separated by six nucleotides. This location prevents thegeneration of any other spacer sequences, such as detection tags andsecondary target sequences, from unligated open circle probes during DNAreplication.

3. Detection Tag Portions

Detection tag portions are part of the spacer region of an open circleprobe. Detection tag portions have sequences matching the sequence ofthe complementary portion of detection probes. These detection tagportions, when amplified during rolling circle replication, result inTS-DNA having detection tag sequences that are complementary to thecomplementary portion of detection probes. If present, there may be one,two, three, or more than three detection tag portions on an OCP. It ispreferred that an OCP have two, three or four detection tag portions.Most preferably, an OCP will have three detection tag portions.Generally, it is preferred that an OCP have 60 detection tag portions orless. There is no fundamental limit to the number of detection tagportions that can be present on an OCP except the size of the OCP. Whenthere are multiple detection tag portions, they may have the samesequence or they may have different sequences, with each differentsequence complementary to a different detection probe. It is preferredthat an OCP contain detection tag portions that have the same sequencesuch that they are all complementary to a single detection probe. Forsome multiplex detection methods, it is preferable that OCPs contain upto six detection tag portions and that the detection tag portions havedifferent sequences such that each of the detection tag portions iscomplementary to a different detection probe. The detection tag portionscan each be any length that supports specific and stable hybridizationbetween the detection tags and the detection probe. For this purpose, alength of 10 to 35 nucleotides is preferred, with a detection tagportion 15 to 20 nucleotides long being most preferred.

4. Secondary Target Sequence Portions

Secondary target sequence portions are part of the spacer region of anopen circle probe. Secondary target sequence portions have sequencesmatching the sequence of target probes of a secondary open circle probe.These secondary target sequence portions, when amplified during rollingcircle replication, result in TS-DNA having secondary target sequencesthat are complementary to target probes of a secondary open circleprobe. If present, there may be one, two, or more than two secondarytarget sequence portions on an OCP. It is preferred that an OCP have oneor two secondary target sequence portions. Most preferably, an OCP willhave one secondary target sequence portion. Generally, it is preferredthat an OCP have 50 secondary target sequence portions or less. There isno fundamental limit to the number of secondary target sequence portionsthat can be present on an OCP except the size of the OCP. When there aremultiple secondary target sequence portions, they may have the samesequence or they may have different sequences, with each differentsequence complementary to a different secondary OCP. It is preferredthat an OCP contain secondary target sequence portions that have thesame sequence such that they are all complementary to a single targetprobe portion of a secondary OCP. The secondary target sequence portionscan each be any length that supports specific and stable hybridizationbetween the secondary target sequence and the target sequence probes ofits cognate OCP. For this purpose, a length of 20 to 70 nucleotides ispreferred, with a secondary target sequence portion 30 to 40 nucleotideslong being most preferred. As used herein, a secondary open circle probeis an open circle probe where the target probe portions match or arecomplementary to secondary target sequences in another open circle probeor an amplification target circle. It is contemplated that a secondaryopen circle probe can itself contain secondary target sequences thatmatch or are complementary to the target probe portions of anothersecondary open circle probe. Secondary open circle probes related toeach other in this manner are referred to herein as nested open circleprobes.

5. Address Tag Portion

The address tag portion is part of either the target probe portions orthe spacer region of an open circle probe. The address tag portion has asequence matching the sequence of the complementary portion of anaddress probe. This address tag portion, when amplified during rollingcircle replication, results in TS-DNA having address tag sequences thatare complementary to the complementary portion of address probes. Ifpresent, there may be one, or more than one, address tag portions on anOCP. It is preferred that an OCP have one or two address tag portions.Most preferably, an OCP will have one address tag portion. Generally, itis preferred that an OCP have 50 address tag portions or less. There isno fundamental limit to the number of address tag portions that can bepresent on an OCP except the size of the OCP. When there are multipleaddress tag portions, they may have the same sequence or they may havedifferent sequences, with each different sequence complementary to adifferent address probe. It is preferred that an OCP contain address tagportions that have the same sequence such that they are allcomplementary to a single address probe. Preferably, the address tagportion overlaps all or a portion of the target probe portions, and allof any intervening gap space (FIG. 6). Most preferably, the address tagportion overlaps all or a portion of both the left and right targetprobe portions. The address tag portion can be any length that supportsspecific and stable hybridization between the address tag and theaddress probe. For this purpose, a length between 10 and 35 nucleotideslong is preferred, with an address tag portion 15 to 20 nucleotides longbeing most preferred.

6. Promoter Portion

The promoter portion corresponds to the sequence of an RNA polymerasepromoter. A promoter portion can be included in an open circle probe sothat transcripts can be generated from TS-DNA. The sequence of anypromoter may be used, but simple promoters for RNA polymerases withoutcomplex requirements are preferred. It is also preferred that thepromoter is not recognized by any RNA polymerase that may be present inthe sample containing the target nucleic acid sequence. Preferably, thepromoter portion corresponds to the sequence of a T7 or SP6 RNApolymerase promoter. The T7 and SP6 RNA polymerases are highly specificfor particular promoter sequences. Other promoter sequences specific forRNA polymerases with this characteristic would also be preferred.Because promoter sequences are generally recognized by specific RNApolymerases, the cognate polymerase for the promoter portion of the OCPshould be used for transcriptional amplification. Numerous promotersequences are known and any promoter specific for a suitable RNApolymerase can be used. The promoter portion can be located anywherewithin the spacer region of an OCP and can be in either orientation.Preferably, the promoter portion is immediately adjacent to the lefttarget probe and is oriented to promote transcription toward the 3' endof the open circle probe. This orientation results in transcripts thatare complementary to TS-DNA, allowing independent detection of TS-DNAand the transcripts, and prevents transcription from interfering withrolling circle replication.

B. Gap Oligonucleotides

Gap oligonucleotides are oligonucleotides that are complementary to allor a part of that portion of a target sequence which covers a gap spacebetween the ends of a hybridized open circle probe. An example of a gapoligonucleotide and its relationship to a target sequence and opencircle probe is shown in FIG. 2. Gap oligonucleotides have a phosphategroup at their 5' ends and a hydroxyl group at their 3' ends. Thisfacilitates ligation of gap oligonucleotides to open circle probes, orto other gap oligonucleotides. The gap space between the ends of ahybridized open circle probe can be filled with a single gapoligonucleotide, or it can be filled with multiple gap oligonucleotides.For example, two 3 nucleotide gap oligonucleotides can be used to fill asix nucleotide gap space, or a three nucleotide gap oligonucleotide anda four nucleotide gap oligonucleotide can be used to fill a sevennucleotide gap space. Gap oligonucleotides are particularly useful fordistinguishing between closely related target sequences. For example,multiple gap oligonucleotides can be used to amplify different allelicvariants of a target sequence. By placing the region of the targetsequence in which the variation occurs in the gap space formed by anopen circle probe, a single open circle probe can be used to amplifyeach of the individual variants by using an appropriate set of gapoligonucleotides.

C. Amplification Target Circles

An amplification target circle (ATC) is a circular single-stranded DNAmolecule, generally containing between 40 to 1000 nucleotides,preferably between about 50 to 150 nucleotides, and most preferablybetween about 50 to 100 nucleotides. Portions of ATCs have specificfunctions making the ATC useful for rolling circle amplification (RCA).These portions are referred to as the primer complement portion, thedetection tag portions, the secondary target sequence portions, theaddress tag portions, and the promoter portion. The primer complementportion is a required element of an amplification target circle.Detection tag portions, secondary target sequence portions, address tagportions, and promoter portions are optional. Generally, anamplification target circle is a single-stranded, circular DNA moleculecomprising a primer complement portion. Those segments of the ATC thatdo not correspond to a specific portion of the ATC can be arbitrarilychosen sequences. It is preferred that ATCs do not have any sequencesthat are self-complementary. It is considered that this condition is metif there are no complementary regions greater than six nucleotides longwithout a mismatch or gap. It is also preferred that ATCs containing apromoter portion do not have any sequences that resemble a transcriptionterminator, such as a run of eight or more thymidine nucleotides.Ligated open circle probes are a type of ATC, and as used herein theterm amplification target circle includes ligated open circle probes. AnATC can be used in the same manner as described herein for OCPs thathave been ligated.

An amplification target circle, when replicated, gives rise to a longDNA molecule containing multiple repeats of sequences complementary tothe amplification target circle. This long DNA molecule is referred toherein as tandem sequences DNA (TS-DNA). TS-DNA contains sequencescomplementary to the primer complement portion and, if present on theamplification target circle, the detection tag portions, the secondarytarget sequence portions, the address tag portions, and the promoterportion. These sequences in the TS-DNA are referred to as primersequences (which match the sequence of the rolling circle replicationprimer), spacer sequences (complementary to the spacer region),detection tags, secondary target sequences, address tags, and promotersequences. Amplification target circles are useful as tags for specificbinding molecules.

D. Rolling Circle Replication Primer

A rolling circle replication primer (RCRP) is an oligonucleotide havingsequence complementary to the primer complement portion of an OCP orATC. This sequence is referred to as the complementary portion of theRCRP. The complementary portion of a RCRP and the cognate primercomplement portion can have any desired sequence so long as they arecomplementary to each other. In general, the sequence of the RCRP can bechosen such that it is not significantly complementary to any otherportion of the OCP or ATC. The complementary portion of a rolling circlereplication primer can be any length that supports specific and stablehybridization between the primer and the primer complement portion.Generally this is 10 to 35 nucleotides long, but is preferably 16 to 20nucleotides long.

It is preferred that rolling circle replication primers also containadditional sequence at the 5' end of the RCRP that is not complementaryto any part of the OCP or ATC. This sequence is referred to as thenon-complementary portion of the RCRP. The non-complementary portion ofthe RCRP, if present, serves to facilitate strand displacement duringDNA replication. The non-complementary portion of a RCRP may be anylength, but is generally 1 to 100 nucleotides long, and preferably 4 to8 nucleotides long. The rolling circle replication primer may alsoinclude modified nucleotides to make it resistant to exonucleasedigestion. For example, the primer can have three or fourphosphorothioate linkages between nucleotides at the 5' end of theprimer. Such nuclease resistant primers allow selective degradation ofexcess unligated OCP and gap oligonucleotides that might otherwiseinterfere with hybridization of detection probes, address probes, andsecondary OCPs to the amplified nucleic acid. A rolling circlereplication primer can be used as the tertiary DNA strand displacementprimer in strand displacement cascade amplification.

E. Detection Labels

To aid in detection and quantitation of nucleic acids amplified usingRCA and RCT, detection labels can be directly incorporated intoamplified nucleic acids or can be coupled to detection molecules. Asused herein, a detection label is any molecule that can be associatedwith amplified nucleic acid, directly or indirectly, and which resultsin a measurable, detectable signal, either directly or indirectly. Manysuch labels for incorporation into nucleic acids or coupling to nucleicacid or antibody probes are known to those of skill in the art. Examplesof detection labels suitable for use in RCA and RCT are radioactiveisotopes, fluorescent molecules, phosphorescent molecules, enzymes,antibodies, and ligands.

Examples of suitable fluorescent labels include fluorescein (FITC),5,6-carboxymethyl fluorescein, Texas red,nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride,rhodamine, 4'-6-diamidino-2-phenylinodole (DAPI), and the cyanine dyesCy3, Cy3.5, Cy5, Cy5.5 and Cy7. Preferred fluorescent labels arefluorescein (5-carboxyfluorescein-N-hydroxysuccinimide ester) andrhodamine (5,6-tetramethyl rhodamine). Preferred fluorescent labels forcombinatorial multicolor coding are FITC and the cyanine dyes Cy3,Cy3.5, Cy5, Cy5.5 and Cy7. The absorption and emission maxima,respectively, for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm;568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm;703 nm) and Cy7 (755 nm; 778 nm), thus allowing their simultaneousdetection. The fluorescent labels can be obtained from a variety ofcommercial sources, including Molecular Probes, Eugene, OR and ResearchOrganics, Cleveland, Ohio.

Labeled nucleotides are preferred form of detection label since they canbe directly incorporated into the products of RCA and RCT duringsynthesis. Examples of detection labels that can be incorporated intoamplified DNA or RNA include nucleotide analogs such as BrdUrd (Hoy andSchimke, Mutation Research 290:217-230 (1993)), BrUTP (Wansick et al.,J. Cell Biology 122:283-293 (1993)) and nucleotides modified with biotin(Langer et al., Proc. Natl. Acad. Sci. USA 78:6633 (1981)) or withsuitable haptens such as digoxygenin (Kerkhof, Anal. Biochem.205:359-364 (1992)). Suitable fluorescence-labeled nucleotides areFluorescein-isothiocyanate-dUTP, Cyanine-3-dUTP and Cyanine-5-dUTP (Yuet al., Nucleic Acids Res., 22:3226-3232 (1994)). A preferred nucleotideanalog detection label for DNA is BrdUrd (BUDR triphosphate, Sigma), anda preferred nucleotide analog detection label for RNA isBiotin-16-uridine-5'-triphosphate (Biotin-16-dUTP, BoehringherMannheim). Fluorescein, Cy3, and Cy5 can be linked to dUTP for directlabelling. Cy3.5 and Cy7 are available as avidin or anti-digoxygeninconjugates for secondary detection of biotin- or digoxygenin-labelledprobes.

Detection labels that are incorporated into amplified nucleic acid, suchas biotin, can be subsequently detected using sensitive methodswell-known in the art. For example, biotin can be detected usingstreptavidin-alkaline phosphatase conjugate (Tropix, Inc.), which isbound to the biotin and subsequently detected by chemiluminescence ofsuitable substrates (for example, chemiluminescent substrate CSPD:disodium, 3-(4-methoxyspiro-[1,2,-dioxetane-3-2'-(5'-chloro)tricyclo[3.3.1.1³,7 ]decane]-4-yl) phenyl phosphate; Tropix, Inc.).

A preferred detection label for use in detection of amplified RNA isacridinium-ester-labeled DNA probe (GenProbe, Inc., as described byArnold et al., Clinical Chemistry 35:1588-1594 (1989)). Anacridinium-ester-labeled detection probe permits the detection ofamplified RNA without washing because unhybridized probe can bedestroyed with alkali (Arnold et al. (1989)).

Molecules that combine two or more of these detection labels are alsoconsidered detection labels. Any of the known detection labels can beused with the disclosed probes, tags, and method to label and detectnucleic acid amplified using the disclosed method. Methods for detectingand measuring signals generated by detection labels are also known tothose of skill in the art. For example, radioactive isotopes can bedetected by scintillation counting or direct visualization; fluorescentmolecules can be detected with fluorescent spectrophotometers;phosphorescent molecules can be detected with a spectrophotometer ordirectly visualized with a camera; enzymes can be detected by detectionor visualization of the product of a reaction catalyzed by the enzyme;antibodies can be detected by detecting a secondary detection labelcoupled to the antibody. Such methods can be used directly in thedisclosed method of amplification and detection. As used herein,detection molecules are molecules which interact with amplified nucleicacid and to which one or more detection labels are coupled.

F. Detection Probes

Detection probes are labeled oligonucleotides having sequencecomplementary to detection tags on TS-DNA or transcripts of TS-DNA. Thecomplementary portion of a detection probe can be any length thatsupports specific and stable hybridization between the detection probeand the detection tag. For this purpose, a length of 10 to 35nucleotides is preferred, with a complementary portion of a detectionprobe 16 to 20 nucleotides long being most preferred. Detection probescan contain any of the detection labels described above. Preferredlabels are biotin and fluorescent molecules. A particularly preferreddetection probe is a molecular beacon. Molecular beacons are detectionprobes labeled with fluorescent moieties where the fluorescent moietiesfluoresce only when the detection probe is hybridized (Tyagi and Kramer,Nature Biotechnology 14:303-308 (1996)). The use of such probeseliminates the need for removal of unhybridized probes prior to labeldetection because the unhybridized detection probes will not produce asignal. This is especially useful in multiplex assays.

A preferred form of detection probe, referred to herein as a collapsingdetection probe, contains two separate complementary portions. Thisallows each detection probe to hybridize to two detection tags inTS-DNA. In this way, the detection probe forms a bridge betweendifferent parts of the TS-DNA. The combined action of numerouscollapsing detection probes hybridizing to TS-DNA will be to form acollapsed network of cross-linked TS-DNA. Collapsed TS-DNA occupies amuch smaller volume than free, extended TS-DNA, and includes whateverdetection label present on the detection probe. This result is a compactand discrete detectable signal for each TS-DNA. Collapsing TS-DNA isuseful both for in situ hybridization applications and for multiplexdetection because it allows detectable signals to be spatially separateeven when closely packed. Collapsing TS-DNA is especially preferred foruse with combinatorial multicolor coding.

TS-DNA collapse can also be accomplished through the use ofligand/ligand binding pairs (such as biotin and avidin) orhapten/antibody pairs. As described in Example 6, a nucleotide analog,BUDR, can be incorporated into TS-DNA during rolling circle replication.When biotinylated antibodies specific for BUDR and avidin are added, across-linked network of TS-DNA forms, bridged by avidin-biotin-antibodyconjugates, and the TS-DNA collapses into a compact structure.Collapsing detection probes and biotin-mediated collapse can also beused together to collapse TS-DNA.

G. Address Probes

An address probe is an oligonucleotide having a sequence complementaryto address tags on TS-DNA or transcripts of TS-DNA. The complementaryportion of an address probe can be any length that supports specific andstable hybridization between the address probe and the address tag. Forthis purpose, a length of 10 to 35 nucleotides is preferred, with acomplementary portion of an address probe 12 to 18 nucleotides longbeing most preferred. Preferably, the complementary portion of anaddress probe is complementary to all or a portion of the target probeportions of an OCP. Most preferably, the complementary portion of anaddress probe is complementary to a portion of either or both of theleft and right target probe portions of an OCP and all or a part of anygap oligonucleotides or gap sequence created in a gap-filling operation(see FIG. 6). Address probe can contain a single complementary portionor multiple complementary portions. Preferably, address probes arecoupled, either directly or via a spacer molecule, to a solid-statesupport. Such a combination of address probe and solid-state support area preferred form of solid-state detector.

H. DNA Strand Displacement Primers

Primers used for secondary DNA strand displacement are referred toherein as DNA strand displacement primers. One form of DNA stranddisplacement primer, referred to herein as a secondary DNA stranddisplacement primer, is an oligonucleotide having sequence matching partof the sequence of an OCP or ATC. This sequence is referred to as thematching portion of the secondary DNA strand displacement primer. Thismatching portion of a secondary DNA strand displacement primer iscomplementary to sequences in TS-DNA. The matching portion of asecondary DNA strand displacement primer may be complementary to anysequence in TS-DNA. However, it is preferred that it not becomplementary TS-DNA sequence matching either the rolling circlereplication primer or a tertiary DNA strand displacement primer, if oneis being used. This prevents hybridization of the primers to each other.The matching portion of a secondary DNA strand displacement primer maybe complementary to all or a portion of the target sequence. In thiscase, it is preferred that the 3' end nucleotides of the secondary DNAstrand displacement primer are complementary to the gap sequence in thetarget sequence. It is most preferred that nucleotide at the 3' end ofthe secondary DNA strand displacement primer falls complementary to thelast nucleotide in the gap sequence of the target sequence, that is, the5' nucleotide in the gap sequence of the target sequence. The matchingportion of a secondary DNA strand displacement primer can be any lengththat supports specific and stable hybridization between the primer andits complement. Generally this is 12 to 35 nucleotides long, but ispreferably 18 to 25 nucleotides long.

It is preferred that secondary DNA strand displacement primers alsocontain additional sequence at their 5' end that does not match any partof the OCP or ATC. This sequence is referred to as the non-matchingportion of the secondary DNA strand displacement primer. Thenon-matching portion of the secondary DNA strand displacement primer, ifpresent, serves to facilitate strand displacement during DNAreplication. The non-matching portion of a secondary DNA stranddisplacement primer may be any length, but is generally 1 to 100nucleotides long, and preferably 4 to 8 nucleotides long.

Another form of DNA strand displacement primer, referred to herein as atertiary DNA strand displacement primer, is an oligonucleotide havingsequence complementary to part of the sequence of an OCP or ATC. Thissequence is referred to as the complementary portion of the tertiary DNAstrand displacement primer. This complementary portion of the tertiaryDNA strand displacement primer matches sequences in TS-DNA. Thecomplementary portion of a tertiary DNA strand displacement primer maybe complementary to any sequence in the OCP or ATC. However, it ispreferred that it not be complementary OCP or ATC sequence matching thesecondary DNA strand displacement primer. This prevents hybridization ofthe primers to each other. Preferably, the complementary portion of thetertiary DNA strand displacement primer has sequence complementary to aportion of the spacer portion of an OCP. The complementary portion of atertiary DNA strand displacement primer can be any length that supportsspecific and stable hybridization between the primer and its complement.Generally this is 12 to 35 nucleotides long, but is preferably 18 to 25nucleotides long. It is preferred that tertiary DNA strand displacementprimers also contain additional sequence at their 5' end that is notcomplementary to any part of the OCP or ATC. This sequence is referredto as the non-complementary portion of the tertiary DNA stranddisplacement primer. The non-complementary portion of the tertiary DNAstrand displacement primer, if present, serves to facilitate stranddisplacement during DNA replication. The non-complementary portion of atertiary DNA strand displacement primer may be any length, but isgenerally 1 to 100 nucleotides long, and preferably 4 to 8 nucleotideslong. A rolling circle replication primer is a preferred form oftertiary DNA strand displacement primer.

DNA strand displacement primers may also include modified nucleotides tomake them resistant to exonuclease digestion. For example, the primercan have three or four phosphorothioate linkages between nucleotides atthe 5' end of the primer. Such nuclease resistant primers allowselective degradation of excess unligated OCP and gap oligonucleotidesthat might otherwise interfere with hybridization of detection probes,address probes, and secondary OCPs to the amplified nucleic acid. DNAstrand displacement primers can be used for secondary DNA stranddisplacement and strand displacement cascade amplification, bothdescribed below.

I. Interrogation Probes

An interrogation probe is an oligonucleotide having a sequencecomplementary to portions of TS-DNA or transcripts of TS-DNA.Interrogation probes are intended for use in primer extension sequencingoperations following rolling circle amplification of an OCP oramplification target circle (for example, USA-SEQ and USA-CAGESEQ).Interrogation probes can be used directly as interrogation primers in aprimer extension sequencing operation, or they can be combined withother interrogation probes or with degenerate probes to forminterrogation primers. As use herein, interrogation primers areoligonucleotides serving as primers for primer extension sequencing. Therelationship of interrogation probes to sequences in OCPs or ATCs (and,therefore, in amplified target sequences) is preferably determined bythe relationship of the interrogation primer (which is formed from theinterrogation probe) to sequences in OCPs or ATCs.

The complementary portion of an interrogation probe can be any lengththat supports hybridization between the interrogation probe and TS-DNA.For this purpose, a length of 10 to 40 nucleotides is preferred, with acomplementary portion of an interrogation probe 15 to 30 nucleotideslong being most preferred. The preferred use of interrogation probes isto form interrogation primers for primer extension sequencing of anamplified target sequence. For this purpose, interrogation probes shouldhybridize to TS-DNA 5' of the portion of the amplified target sequencesthat are to be sequenced.

For primer extension sequencing operations (for example, USA-CAGESEQ),it is preferred that a nested set of interrogation probes are designedto hybridize just 5' to a region of amplified target sequence for whichthe sequence is to be determined. Thus, for example, a set ofinterrogation probes can be designed where each probe is complementaryto a 20 nucleotide region of the target sequence with each 20 nucleotideregion offset from the previous region by one nucleotide. This preferredrelationship can be illustrated as follows: Probe 1 TCTCGACATCTAACGATCGAProbe 2 CTCGACATCTAACGATCGAT Probe 3 TCGACATCTAACGATCGATC Probe 4CGACATCTAACGATCGATCC Probe 5 GACATCTAACGATCGATCCT ||||||.vertline.||||||.ver tline.|||||| TargetTAGAGCTGTAGATTGCTAGCTAGGATCACACACACACACACA

Probe 1 is nucleotides 76 to 95 of SEQ ID NO:25, probe 2 is nucleotides77 to 96 of SEQ ID NO:25, probe 3 is nucleotides 78 to 97 of SEQ IDNO:25, probe 4 is nucleotides 79 to 98 of SEQ ID NO:25, probe 5 isnucleotides 80 to 99 of SEQ ID NO:25, and the target (shown 3' to 5') isnucleotides 19 to 60 of SEQ ID NO:19. It is preferred that the number ofinterrogation probes in such a nested set be equal to the length of thedegenerate probes used in the primer extension sequencing operation.

It is also preferred that the 3' hydroxyl of interrogation probes bereversibly blocked in order to prevent unwanted ligation to otheroligonucleotides. Such blocked probes allow controlled ligation ofadditional probes, such as degenerate probes, to an interrogation probe.For example, USA-CAGESEQ, a form of degenerate probe primer extensionsequencing, makes use of reversibly blocked interrogation probes toallow sequential, and controlled, addition of degenerate probes tointerrogation probes. Any of the known means of reversibly blocking3'-hydroxyls in oligonucleotides can be used to produce blockedinterrogation probes. Preferred forms of reversible blocking elementsare the cage structures described below. Caged oligonucleotides usefulas blocked interrogation probes are described below.

J. Degenerate Probes

Degenerate probes are oligonucleotides intended for use in primerextension sequencing operations following rolling circle amplificationof an OCP or amplification target circle (for example, USA-SEQ andUSA-CAGESEQ). Degenerate probes are combined with interrogation probesto form interrogation primers. This is accomplished by hybridizing aninterrogation probe and degenerate primers to TS-DNA and ligatingtogether the interrogation probe and whichever degenerate probe that ishybridized adjacent to the interrogation probe. For this purpose, it ispreferred that a full set of degenerate probes be used together. Thisensures that at least one of the degenerate probes will be complementaryto the portion of TS-DNA immediately adjacent to a hybridizedinterrogation probe. This preferred relationship can be illustrated asfollows: Interrogation probe Degenerate probe GACATCTAACGATCGATCCTAGTGT||||||.vertline.|||||||.vertline.||||||.vertline.|||TAGAGCTGTAGATTGCTAGCTAGGATCACACACACACACACA Target

The interrogation probe and degenerate probe together representnucleotides 80 to 104 of SEQ ID NO:25, and the target (shown 3' to 5')is nucleotides 19 to 60 of SEQ ID NO:19. The underlined sequencerepresents the degenerate probe which is ligated to the interrogationprobe (non-underlined portion of the top sequence).

It is preferred that a full set of degenerate probes be used in primerextension sequencing operations involving degenerate probes. As usedherein, a full set of degenerate probes refers to a set ofoligonucleotides, all of the same length, where every possiblenucleotide sequence is represented. The number of such probes isdescribed by the formula 4^(N) where 4 represents the four types ofnucleotides found in DNA (or in RNA) and N is the length of theoligonucleotides in the set. Thus, a full set of degenerate probes threenucleotides in length would include 64 different oligonucleotides, afull set of degenerate probes four nucleotides in length would include256 different oligonucleotides, and a full set of degenerate probes fivenucleotides in length would include 1024 different oligonucleotides. Itis preferred that the number of interrogation probes in such a nestedset be equal to the length of the degenerate probes used in degenerateprobe primer extension sequencing. Sets of degenerate probes can be usedwith a single interrogation probe or with sets of interrogation probes.It is preferred that such sets of interrogation probes represent anested set as described above.

In a primer extension operation, only one of the degenerate probes in aset of degenerate probes will hybridize adjacent to a giveninterrogation probe hybridized to an amplified target sequence. Thenucleotide sequence adjacent to (that is, 3' of) the region of thetarget sequence hybridized to the interrogation probe determines whichdegenerate probe will hybridize. Only degenerate probes hybridizedimmediately adjacent to the interrogation probe should be ligated to theinterrogation probe. For this reason, it is preferred that the region ofthe target sequence to be sequenced is adjacent to the region hybridizedto the interrogation probe. Preferably, this region is a gap sequence inTS-DNA (representing all or a portion of a filled gap space). The use ofgap-filling ligation allows the presence of gap sequences in TS-DNArepresenting a potential, expected, or known region of sequencevariability in the target nucleic aid which is amplified in RCA.

Degenerate probes can be combined with interrogation probes or withother degenerate probes to form interrogation primers. As used herein,interrogation primers are oligonucleotides serving as primers for primerextension sequencing.

It is also preferred that the 3' hydroxyl of degenerate probes bereversibly blocked in order to prevent unwanted ligation to otheroligonucleotides. Such blocked probes allow controlled ligation ofadditional degenerate probes to a degenerate probe. For example,USA-CAGESEQ makes use of reversibly blocked degenerate probes to allowsequential, and controlled, addition of the degenerate probes tointerrogation probes. Any of the known means of reversibly blocking3'-hydroxyls in oligonucleotides can be used to produce blockeddegenerate probes. Preferred forms of reversible blocking elements arethe cage structures described below. Caged oligonucleotides useful asblocked degenerate probes are described below.

Where a nested set of interrogation probes are used, they can be used ina set of primer extension sequencing operations to determine theidentity of adjacent nucleotides. Using the set of interrogation probesillustrated above, and a full set of pentamer degenerate probes, thehighlighted nucleotides in the target sequence can be identified where asingle degenerate probe is ligated to each of the interrogation probes:Probe 1 TCTCGACATCTAACGATCGA Probe 2 CTCGACATCTAACGATCGAT Probe 3TCGACATCTAACGATCGATC Probe 4 CGACATCTAACGATCGATCC Probe 5GACATCTAACGATCGATCCT ||||||.vertli ne.||||||.ver tline.|||||| TargetTAGAGCTGTAGATTGCTAGCTAGGATCACACACACACACACA

Probe 1 is nucleotides 76 to 95 of SEQ ID NO:25, probe 2 is nucleotides77 to 96 of SEQ ID NO:25, probe 3 is nucleotides 78 to 97 of SEQ IDNO:25, probe 4 is nucleotides 79 to 98 of SEQ ID NO:25, probe 5 isnucleotides 80 to 99 of SEQ ID NO:25, and the target (shown 3' to 5') isnucleotides 19 to 60 of SEQ ID NO:19. The highlighted nucleotidesrepresent the nucleotides adjacent to (that is, 3' of) the interrogationprimers formed by the ligation of the interrogation probes and thedegenerate primers. The identity of additional nucleotides can bedetermined by ligating additional degenerate probes to the degenerateprobes already ligated to the interrogation probes. This process isillustrated Example 10. It is preferred that the length of thedegenerate probes be equal to the number of interrogation probes in anested set.

K. Interrogation Primers

An interrogation primer is an oligonucleotide having a sequencecomplementary to portions of TS-DNA or transcripts of TS-DNA.Interrogation primers are intended for use in primer extensionsequencing operations following rolling circle amplification of anamplification target circle (for example, USA-SEQ and USA-CAGESEQ).Preferably, an interrogation primer is complementary to a portion of thetarget sequences in TS-DNA representing all or a portion of the lefttarget probe portion of an OCP. For use with secondary TS-DNA, it ispreferred that interrogation primers are complementary to a portion ofthe target sequences in secondary TS-DNA representing the right targetprobe portion of an OCP. Such interrogation primers are also preferablycomplementary to a portion of the spacer region adjacent to the lefttarget probe portion. Thus, preferred interrogation primers arecomplementary to a contiguous segment of TS-DNA representing the 5' endof the OCP. This preferred relationship allows primer extensionsequencing of gap sequences in TS-DNA. An example of this preferredrelationship between interrogation primers and an OCP is shown in FIG.15. An interrogation probe can, however, be complementary to any desiredsequence in amplified nucleic acid.

In general, interrogation primers can be an unligated interrogationprobe, a combination of two or more interrogation probes (that is,interrogation probes ligated together), or a combination of one or moreinterrogation probes and one or more degenerate probes (that is,interrogation probes and degenerate probes ligated together). Thus,interrogation probes can be used directly as interrogation primers in aprimer extension sequencing operation, or they can be combined withother interrogation probes or with degenerate probes to forminterrogation primers. As use herein, interrogation primers areoligonucleotides serving as primers for primer extension sequencing.Where an interrogation primer is made from probes with blocked3'-hydroxyls, and the resulting interrogation primer is blocked, theblock must be removed prior to the primer extension operation.

The complementary portion of an interrogation primer can be any lengththat supports specific and stable hybridization between theinterrogation primer and TS-DNA. For this purpose, a length of 10 to 40nucleotides is preferred, with a complementary portion of aninterrogation primer 15 to 30 nucleotides long being most preferred. Thepreferred use of interrogation primers as primers in primer extensionsequencing of an amplified target sequence. For this purpose,interrogation primers should hybridize to TS-DNA 5' of the portion ofthe amplified target sequences that are to be sequenced. It is preferredthat the portion of the amplified target sequences that are to besequenced represent gap sequences. Such gap sequences preferablycollectively represent known, expected, or potential sequence variantspresent in the portion of the target nucleic acid opposite the gap spaceformed when an OCP hybridizes to the target nucleic acid. For thispurpose, it is preferred that the gap space is filled by DNA polymerasein a gap-filling ligation operation.

L. Caged Oligonucleotides

Caged oligonucleotides are oligonucleotides having a caged nucleotide attheir 3' end. The cage structure is a removable blocking group whichprevents the 3' hydroxyl from participating in nucleotide addition andligation reactions. Caged oligonucleotides are useful as primers andprobes as described above for use in the amplification, detection, andsequencing operations disclosed herein. Many cage structures are known.A preferred form of cage structure are photolabile structures whichallow their removal by exposure to light. Examples of cage structuresuseful for reversibly blocking the 3' end of oligonucleotides aredescribed by Metzker et al., Nucleic Acids Research 22:4259-4267 (1994),Burgess and Jacutin, Am. Chem Soc. Abstracts volume 221, abstract 281(1996), Zehavi et al., J. Organic Chem. 37:2281-2288 (1972), Kaplan etal., Biochem. 17:1929-1935 (1978), and McCray et al., Proc. Natl. Acad.Sci. USA 77:7237-7241 (1980). Preferred forms of caged nucleotides foruse in caged oligonucleotides are described by Metzker et al. A mostpreferred cage structures is a 3'-O-(2-nitrobenzyl) group, which islabile upon exposure to ultraviolet light (Pillai, Synthesis 1-26(1980)). Removal of this cage structure is preferably accomplished byilluminating the material containing the caged nucleotide with longwavelength ultraviolet light (preferably 354 nm) using atransilluminator for 3 to 10 minutes.

Disclosed and known cage structures can be incorporated intooligonucleotides by adapting known and established oligonucleotidesynthesis methodology (described below) to use protected cagednucleotides or by adding the cage structure following oligonucleotidesynthesis.

As described above, caged oligonucleotides can be used as interrogationprobes or degenerate probes. Caged oligonucleotides can also be used asreplication primers, such as rolling circle replication primers, eitherfor the entire population of, or a portion of, the primers in anamplification reaction. This allows the pool of functional (that is,extendable) primers to be increased at a specified point in the reactionor amplification operation. For example, when using different rollingcircle replication primers to produce different lengths of TS-DNA (seeSection II B below), one of the rolling circle replication primers canbe a caged oligonucleotide.

M. Peptide Nucleic Acid Clamps

Peptide nucleic acids (PNA) are a modified form of nucleic acid having apeptide backbone. Peptide nucleic acids form extremely stable hybridswith DNA (Hanvey et al., Science 258:1481-1485 (1992); Nielsen et al.,Anticancer Drug Des. 8:53-63 (1993)), and have been used as specificblockers of PCR reactions (Orum et al., Nucleic Acids Res., 21:5332-5336(1993)). PNA clamps are peptide nucleic acids complementary to sequencesin both the left target probe portion and right target probe portion ofan OCP, but not to the sequence of any gap oligonucleotides or filledgap space in the ligated OCP. Thus, a PNA clamp can hybridize only tothe ligated junction of OCPs that have been illegitimately ligated, thatis, ligated in a non-target-directed manner. The PNA clamp can be anylength that supports specific and stable hybridization between the clampand its complement. Generally this is 7 to 12 nucleotides long, but ispreferably 8 to 10 nucleotides long. PNA clamps can be used to reducebackground signals in rolling circle amplifications by preventingreplication of illegitimately ligated OCPs.

N. Oligonucleotide Synthesis

Open circle probes, gap oligonucleotides, rolling circle replicationprimers, detection probes, address probes, amplification target circles,DNA strand displacement primers, and any other oligonucleotides can besynthesized using established oligonucleotide synthesis methods. Methodsto produce or synthesize oligonucleotides are well known in the art.Such methods can range from standard enzymatic digestion followed bynucleotide fragment isolation (see for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) Chapters 5, 6) topurely synthetic methods, for example, by the cyanoethyl phosphoramiditemethod using a Milligen or Beckman System 1Plus DNA synthesizer (forexample, Model 8700 automated synthesizer of Milligen-Biosearch,Burlington, Mass. or ABI Model 380B). Synthetic methods useful formaking oligonucleotides are also described by Ikuta et al., Ann. Rev.Biochem. 53:323-356 (1984), (phosphotriester and phosphite-triestermethods), and Narang et al., Methods Enzymol., 65:610-620 (1980),(phosphotriester method). Protein nucleic acid molecules can be madeusing known methods such as those described by Nielsen et al.,Bioconjug. Chem. 5:3-7 (1994).

Many of the oligonucleotides described herein are designed to becomplementary to certain portions of other oligonucleotides or nucleicacids such that stable hybrids can be formed between them. The stabilityof these hybrids can be calculated using known methods such as thosedescribed in Lesnick and Freier, Biochemistry 34:10807-10815 (1995),McGraw et al., Biotechniques 8:674-678 (1990), and Rychlik et al.,Nucleic Acids Res. 18:6409-6412 (1990).

O. Solid-State Detectors

Solid-state detectors are solid-state substrates or supports to whichaddress probes or detection molecules have been coupled. A preferredform of solid-state detector is an array detector. An array detector isa solid-state detector to which multiple different address probes ordetection molecules have been coupled in an array, grid, or otherorganized pattern.

Solid-state substrates for use in solid-state detectors can include anysolid material to which oligonucleotides can be coupled. This includesmaterials such as acrylamide, cellulose, nitrocellulose, glass,polystyrene, polyethylene vinyl acetate, polypropylene,polymethacrylate, polyethylene, polyethylene oxide, glass,polysilicates, polycarbonates, teflon, fluorocarbons, nylon, siliconrubber, polyanhydrides, polyglycolic acid, polylactic acid,polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans, andpolyamino acids. Solid-state substrates can have any useful formincluding thin films or membranes, beads, bottles, dishes, fibers, wovenfibers, shaped polymers, particles and microparticles. A preferred formfor a solid-state substrate is a microtiter dish. The most preferredform of microtiter dish is the standard 96-well type.

Address probes immobilized on a solid-state substrate allow capture ofthe products of RCA and RCT on a solid-state detector. Such captureprovides a convenient means of washing away reaction components thatmight interfere with subsequent detection steps. By attaching differentaddress probes to different regions of a solid-state detector, differentRCA or RCT products can be captured at different, and thereforediagnostic, locations on the solid-state detector. For example, in amicrotiter plate multiplex assay, address probes specific for up to 96different TS-DNAs (each amplified via a different target sequence) canbe immobilized on a microtiter plate, each in a different well. Captureand detection will occur only in those wells corresponding to TS-DNAsfor which the corresponding target sequences were present in a sample.

Methods for immobilization of oligonucleotides to solid-state substratesare well established. Oligonucleotides, including address probes anddetection probes, can be coupled to substrates using establishedcoupling methods. For example, suitable attachment methods are describedby Pease et al., Proc. Natl. Acad. Sci. USA 91(11):5022-5026 (1994), andKhrapko et al., Mol Biol (Mosk) (USSR) 25:718-730 (1991). A method forimmobilization of 3'-amine oligonucleotides on casein-coated slides isdescribed by Stimpson et al., Proc. Natl. Acad. Sci. USA 92:6379-6383(1995). A preferred method of attaching oligonucleotides to solid-statesubstrates is described by Guo et al., Nucleic Acids Res. 22:5456-5465(1994).

Some solid-state detectors useful in RCA and RCT assays have detectionantibodies attached to a solid-state substrate. Such antibodies can bespecific for a molecule of interest. Captured molecules of interest canthen be detected by binding of a second, reporter antibody, followed byRCA or RCT. Such a use of antibodies in a solid-state detector allowsRCA assays to be developed for the detection of any molecule for whichantibodies can be generated. Methods for immobilizing antibodies tosolid-state substrates are well established. Immobilization can beaccomplished by attachment, for example, to aminated surfaces,carboxylated surfaces or hydroxylated surfaces using standardimmobilization chemistries. Examples of attachment agents are cyanogenbromide, succinimide, aldehydes, tosyl chloride, avidin-biotin,photocrosslinkable agents, epoxides and maleimides. A preferredattachment agent is glutaraldehyde. These and other attachment agents,as well as methods for their use in attachment, are described in Proteinimmobilization: fundamentals and applications, Richard F. Taylor, ed.(M. Dekker, New York, 1991), Johnstone and Thorpe, Immunochemistry InPractice (Blackwell Scientific Publications, Oxford, England, 1987)pages 209-216 and 241-242, and Immobilized Affinity Ligands, Craig T.Hermanson et al., eds. (Academic Press, New York, 1992). Antibodies canbe attached to a substrate by chemically cross-linking a free aminogroup on the antibody to reactive side groups present within thesolid-state substrate. For example, antibodies may be chemicallycross-linked to a substrate that contains free amino or carboxyl groupsusing glutaraldehyde or carbodiimides as cross-linker agents. In thismethod, aqueous solutions containing free antibodies are incubated withthe solid-state substrate in the presence of glutaraldehyde orcarbodiimide. For crosslinking with glutaraldehyde the reactants can beincubated with 2% glutaraldehyde by volume in a buffered solution suchas 0.1 M sodium cacodylate at pH 7.4. Other standard immobilizationchemistries are known by those of skill in the art.

P. Solid-State Samples

Solid-state samples are solid-state substrates or supports to whichtarget molecules or target sequences have been coupled or adhered.Target molecules or target sequences are preferably delivered in atarget sample or assay sample. A preferred form of solid-state sample isan array sample. An array sample is a solid-state sample to whichmultiple different target samples or assay samples have been coupled oradhered in an array, grid, or other organized pattern.

Solid-state substrates for use in solid-state samples can include anysolid material to which target molecules or target sequences can becoupled or adhered. This includes materials such as acrylamide,cellulose, nitrocellulose, glass, polystyrene, polyethylene vinylacetate, polypropylene, polymethacrylate, polyethylene, polyethyleneoxide, glass, polysilicates, polycarbonates, teflon, fluorocarbons,nylon, silicon rubber, polyanhydrides, polyglycolic acid, polylacticacid, polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans,and polyamino acids. Solid-state substrates can have any useful formincluding thin films or membranes, beads, bottles, dishes, slides,fibers, woven fibers, shaped polymers, particles and microparticles.Preferred forms for a solid-state substrate are microtiter dishes andglass slides. The most preferred form of microtiter dish is the standard96-well type.

Target molecules and target sequences immobilized on a solid-statesubstrate allow formation of target-specific TS-DNA localized on thesolid-state substrate. Such localization provides a convenient means ofwashing away reaction components that might interfere with subsequentdetection steps, and a convenient way of assaying multiple differentsamples simultaneously. Diagnostic TS-DNA can be independently formed ateach site where a different sample is adhered. For immobilization oftarget sequences or other oligonucleotide molecules to form asolid-state sample, the methods described above for can be used. Wherethe target molecule is a protein, the protein can be immobilized on asolid-state substrate generally as described above for theimmobilization of antibodies.

A preferred form of solid-state substrate is a glass slide to which upto 256 separate target or assay samples have been adhered as an array ofsmall dots. Each dot is preferably from 0.1 to 2.5 mm in diameter, andmost preferably around 2.5 mm in diameter. Such microarrays can befabricated, for example, using the method described by Schena et al.,Science 270:487-470 (1995). Briefly, microarrays can be fabricated onpoly-L-lysine-coated microscope slides (Sigma) with an arraying machinefitted with one printing tip. The tip is loaded with 1 μl of a DNAsample (0.5 mg/ml) from, for example, 96-well microtiter plates anddeposited ˜0.005 μl per slide on multiple slides at the desired spacing.The printed slides can then be rehydrated for 2 hours in a humidchamber, snap-dried at 100° C. for 1 minute, rinsed in 0.1% SDS, andtreated with 0.05% succinic anhydride prepared in buffer consisting of50% 1-methyl-2-pyrrolidinone and 50% boric acid. The DNA on the slidescan then be denatured in, for example, distilled water for 2 minutes at90° C. immediately before use. Microarray solid-state samples canscanned with, for example, a laser fluorescent scanner with acomputer-controlled XY stage and a microscope objective. A mixed gas,multiline laser allows sequential excitation of multiple fluorophores.

Q. Reporter Binding Agents

A reporter binding agent is a specific binding molecule coupled ortethered to an oligonucleotide. The specific binding molecule isreferred to as the affinity portion of the reporter binding agent andthe oligonucleotide is referred to as the oligonucleotide portion of thereporter binding agent. As used herein, a specific binding molecule is amolecule that interacts specifically with a particular molecule ormoiety. The molecule or moiety that interacts specifically with aspecific binding molecule is referred to herein as a target molecule. Itis to be understood that the term target molecule refers to bothseparate molecules and to portions of molecules, such as an epitope of aprotein, that interacts specifically with a specific binding molecule.Antibodies, either member of a receptor/ligand pair, and other moleculeswith specific binding affinities are examples of specific bindingmolecules, useful as the affinity portion of a reporter bindingmolecule. A reporter binding molecule with an affinity portion which isan antibody is referred to herein as a reporter antibody. By tetheringan amplification target circle or coupling a target sequence to suchspecific binding molecules, binding of a specific binding molecule toits specific target can be detected by amplifying the ATC or targetsequence with rolling circle amplification. This amplification allowssensitive detection of a very small number of bound specific bindingmolecules. A reporter binding molecule that interacts specifically witha particular target molecule is said to be specific for that targetmolecule. For example, a reporter binding molecule with an affinityportion which is an antibody that binds to a particular antigen is saidto be specific for that antigen. The antigen is the target molecule.Reporter binding agents are also referred to herein as reporter bindingmolecules. FIGS. 25, 26, 27, 28, and 29 illustrate examples of severalpreferred types of reporter binding molecules and their use. FIG. 29illustrates a reporter binding molecule using an antibody as theaffinity portion.

A special form of reporter binding molecule, referred to herein as areporter binding probe, has an oligonucleotide or oligonucleotidederivative as the specific binding molecule. Reporter binding probes aredesigned for and used to detect specific nucleic acid sequences. Thus,the target molecule for reporter binding probes are nucleic acidsequences. The target molecule for a reporter binding probe can be anucleotide sequence within a larger nucleic acid molecule. It is to beunderstood that the term reporter binding molecule encompasses reporterbinding probes. The specific binding molecule of a reporter bindingprobe can be any length that supports specific and stable hybridizationbetween the reporter binding probe and the target molecule. For thispurpose, a length of 10 to 40 nucleotides is preferred, with a specificbinding molecule of a reporter binding probe 16 to 25 nucleotides longbeing most preferred. It is preferred that the specific binding moleculeof a reporter binding probe is peptide nucleic acid. As described above,peptide nucleic acid forms a stable hybrid with DNA. This allows areporter binding probe with a peptide nucleic acid specific bindingmolecule to remain firmly adhered to the target sequence duringsubsequent amplification and detection operations. This useful effectcan also be obtained with reporter binding probes with oligonucleotidespecific binding molecules by making use of the triple helix chemicalbonding technology described by Gasparro et al., Nucleic Acids Res. 199422(14):2845-2852 (1994). Briefly, the affinity portion of a reporterbinding probe is designed to form a triple helix when hybridized to atarget sequence. This is accomplished generally as known, preferably byselecting either a primarily homopurine or primarily homopyrimidinetarget sequence. The matching oligonucleotide sequence which constitutesthe affinity portion of the reporter binding probe will be complementaryto the selected target sequence and thus be primarily homopyrimidine orprimarily homopurine, respectively. The reporter binding probe(corresponding to the triple helix probe described by Gasparro et al.)contains a chemically linked psoralen derivative. Upon hybridization ofthe reporter binding probe to a target sequence, a triple helix forms.By exposing the triple helix to low wavelength ultraviolet radiation,the psoralen derivative mediates cross-linking of the probe to thetarget sequence. FIGS. 25, 26, 27, and 28 illustrate examples ofreporter binding molecules that are reporter binding probes.

The specific binding molecule in a reporter binding probe can also be abipartite DNA molecule, such as ligatable DNA probes adapted from thosedescribed by Landegren et al., Science 241:1077-1080 (1988). When usingsuch a probe, the affinity portion of the probe is assembled bytarget-mediated ligation of two oligonucleotide portions which hybridizeto adjacent regions of a target nucleic acid. Thus, the components usedto form the affinity portion of such reporter binding probes are atruncated reporter binding probe (with a truncated affinity portionwhich hybridizes to part of the target sequence) and a ligation probewhich hybridizes to an adjacent part of the target sequence such that itcan be ligated to the truncated reporter binding probe. The ligationprobe can also be separated from (that is, not adjacent to) thetruncated reporter binding probe when both are hybridized to the targetsequence. The resulting space between them can then be filled by asecond ligation probe or by gap-filling synthesis. For use in thedisclosed methods, it is preferred that the truncated affinity portionbe long enough to allow target-mediated ligation but short enough to, inthe absence of ligation to the ligation probe, prevent stablehybridization of the truncated reporter binding probe to the targetsequence during the subsequent amplification operation. For thispurpose, a specific step designed to eliminate hybrids between thetarget sequence and unligated truncated reporter binding probes can beused following the ligation operation.

In one embodiment, the oligonucleotide portion of a reporter bindingagent includes a sequence, referred to as a target sequence, that servesas a target sequence for an OCP. The sequence of the target sequence canbe arbitrarily chosen. In a multiplex assay using multiple reporterbinding agents, it is preferred that the target sequence for eachreporter binding agent be substantially different to limit thepossibility of non-specific target detection. Alternatively, it may bedesirable in some multiplex assays, to use target sequences with relatedsequences. By using different, unique gap oligonucleotides to filldifferent gap spaces, such assays can use one or a few OCPs to amplifyand detect a larger number of target sequences. The oligonucleotideportion can be coupled to the affinity portion by any of severalestablished coupling reactions. For example, Hendrickson et al., NucleicAcids Res., 23(3):522-529 (1995) describes a suitable method forcoupling oligonucleotides to antibodies.

In another embodiment, the oligonucleotide portion of a reporter bindingagent includes a sequence, referred to as a rolling circle replicationprimer sequence, that serves as a rolling circle replication primer foran ATC. This allows rolling circle replication of an added ATC where theresulting TS-DNA is coupled to the reporter binding agent. Because ofthis, the TS-DNA will be effectively immobilized at the site of thetarget molecule. Preferably, the immobilized TS-DNA can then becollapsed in situ prior to detection. The sequence of the rolling circlereplication primer sequence can be arbitrarily chosen. In a multiplexassay using multiple reporter binding agents, it is preferred that therolling circle replication primer sequence for each reporter bindingagent be substantially different to limit the possibility ofnon-specific target detection. Alternatively, it may be desirable insome multiplex assays, to use rolling circle replication primersequences with related sequences. Such assays can use one or a few ATCsto detect a larger number of target molecules. When the oligonucleotideportion of a reporter binding agent is used as a rolling circlereplication primer, the oligonucleotide portion can be any length thatsupports specific and stable hybridization between the oligonucleotideportion and the primer complement portion of an amplification targetcircle. Generally this is 10 to 35 nucleotides long, but is preferably16 to 20 nucleotides long. FIGS. 25, 26, 27, 28, and 29 illustrateexamples of reporter binding molecules in which the oligonucleotideportion is a rolling circle replication primer.

In another embodiment, the oligonucleotide portion of a reporter bindingagent can include an amplification target circle which serves as atemplate for rolling circle replication. In a multiplex assay usingmultiple reporter binding agents, it is preferred that address tagportions and detection tag portions of the ATC comprising theoligonucleotide portion of each reporter binding agent be substantiallydifferent to unique detection of each reporter binding agent. It isdesirable, however, to use the same primer complement portion in all ofthe ATCs used in a multiplex assay. The ATC is tethered to the specificbinding molecule by looping the ATC around a tether loop. This allowsthe ATC to rotate freely during rolling circle replication whileremaining coupled to the affinity portion. The tether loop can be anymaterial that can form a loop and be coupled to a specific bindingmolecule. Linear polymers are a preferred material for tether loops.

A preferred method of producing a reporter binding agent with a tetheredATC is to form the tether loop by ligating the ends of oligonucleotidescoupled to a specific binding molecule around an ATC. Oligonucleotidescan be coupled to specific binding molecules using known techniques. Forexample, Hendrickson et al. (1995), describes a suitable method forcoupling oligonucleotides to antibodies. This method is generally usefulfor coupling oligonucleotides to any protein. To allow ligation,oligonucleotides comprising the two halves of the tether loop should becoupled to the specific binding molecule in opposite orientations suchthat the free end of one is the 5' end and the free end of the other isthe 3' end. Ligation of the ends of the tether oligonucleotides can bemediated by hybridization of the ends of the tether oligonucleotides toadjacent sequences in the ATC to be tethered. In this way, the ends ofthe tether oligonucleotides are analogous to the target probe portionsof an open circle probe, with the ATC containing the target sequence.

Another preferred method of producing a reporter binding agent with atethered ATC is to ligate an open circle probe while hybridized to anoligonucleotide tether loop on a specific binding molecule. This isanalogous to the ligation operation of LM-RCA. In this case, the targetsequence is part of an oligonucleotide with both ends coupled to aspecific binding molecule. In this method, both ends of a single tetheroligonucleotide are coupled to a specific binding molecule. This can beaccomplished using known coupling techniques as described above.

The ends of tether loops can be coupled to any specific binding moleculewith functional groups that can be derivatized with suitable activatinggroups. When the specific binding molecule is a protein, or a moleculewith similar functional groups, coupling of tether ends can beaccomplished using known methods of protein attachment. Many suchmethods are described in Protein immobilization: fundamentals andapplications Richard F. Taylor, ed. (M. Dekker, New York, 1991).

Antibodies useful as the affinity portion of reporter binding agents,can be obtained commercially or produced using well established methods.For example, Johnstone and Thorpe, on pages 30-85, describe generalmethods useful for producing both polyclonal and monoclonal antibodies.The entire book describes many general techniques and principles for theuse of antibodies in assay systems.

R. DNA Ligases

Any DNA ligase is suitable for use in the disclosed amplificationmethod. Preferred ligases are those that preferentially formphosphodiester bonds at nicks in double-stranded DNA. That is, ligasesthat fail to ligate the free ends of single-stranded DNA at asignificant rate are preferred. Thermostable ligases are especiallypreferred. Many suitable ligases are known, such as T4 DNA ligase (Daviset al., Advanced Bacterial Genetics--A Manual for Genetic Engineering(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1980)), E.coli DNA ligase (Panasnko et al., J. Biol. Chem. 253:4590-4592 (1978)),AMPLIGASE® (Kalin et al., Mutat. Res., 283(2):119-123 (1992); Winn-Deenet al., Mol Cell Probes (England) 7(3):179-186 (1993)), Taq DNA ligase(Barany, Proc. Natl. Acad. Sci. USA 88:189-193 (1991), Thermusthermophilus DNA ligase (Abbott Laboratories), Thermus scotoductus DNAligase and Rhodothermus marinus DNA ligase (Thorbjarnardottir et al.,Gene 151:177-180 (1995)). T4 DNA ligase is preferred for ligationsinvolving RNA target sequences due to its ability to ligate DNA endsinvolved in DNA:RNA hybrids (Hsuih et al., Quantitative detection of HCVRNA using novel ligation-dependent polymerase chain reaction, AmericanAssociation for the Study of Liver Diseases (Chicago, Ill., Nov. 3-7,1995)).

The frequency of non-target-directed ligation catalyzed by a ligase canbe determined as follows. LM-RCA is performed with an open circle probeand a gap oligonucleotide in the presence of a target sequence.Non-targeted-directed ligation products can then be detected by using anaddress probe specific for the open circle probe ligated without the gapoligonucleotide to capture TS-DNA from such ligated probes. Targetdirected ligation products can be detected by using an address probespecific for the open circle probe ligated with the gap oligonucleotide.By using a solid-state detector with regions containing each of theseaddress probes, both target directed and non-target-directed ligationproducts can be detected and quantitated. The ratio of target-directedand non-target-directed TS-DNA produced provides a measure of thespecificity of the ligation operation. Target-directed ligation can alsobe assessed as discussed in Barany (1991).

S. DNA Polymerases

DNA polymerases useful in the rolling circle replication step of RCAmust perform rolling circle replication of primed single-strandedcircles. Such polymerases are referred to herein as rolling circle DNApolymerases. For rolling circle replication, it is preferred that a DNApolymerase be capable of displacing the strand complementary to thetemplate strand, termed strand displacement, and lack a 5' to 3'exonuclease activity. Strand displacement is necessary to result insynthesis of multiple tandem copies of the ligated OCP. A 5' to 3'exonuclease activity, if present, might result in the destruction of thesynthesized strand. It is also preferred that DNA polymerases for use inthe disclosed method are highly processive. The suitability of a DNApolymerase for use in the disclosed method can be readily determined byassessing its ability to carry out rolling circle replication. Preferredrolling circle DNA polymerases are bacteriophage φ29 DNA polymerase(U.S. Pat. Nos. 5,198,543 and 5,001,050 to Blanco et al.), phage M2 DNApolymerase (Matsumoto et al., Gene 84:247 (1989)), phage φPRD1 DNApolymerase (Jung et al., Proc. Natl. Acad. Sci. USA 84:8287 (1987)),VENT® DNA polymerase (Kong et al., J. Biol. Chem. 268:1965-1975 (1993)),Klenow fragment of DNA polymerase I (Jacobsen et al., Eur. J. Biochem.45:623-627 (1974)), T5 DNA polymerase (Chatterjee et al., Gene 97:13-19(1991)), PRD1 DNA polymerase (Zhu and Ito, Biochim. Biophys. Acta.1219:267-276 (1994)), modified T7 DNA polymerase (Tabor and Richardson,J. Biol. Chem. 262:15330-15333 (1987); Tabor and Richardson, J. Biol.Chem. 264:6447-6458 (1989); Sequenase™ (U.S. Biochemicals)), and T4 DNApolymerase holoenzyme (Kaboord and Benkovic, Curr. Biol. 5:149-157(1995)). φ29 DNA polymerase is most preferred.

Strand displacement can be facilitated through the use of a stranddisplacement factor, such as helicase. It is considered that any DNApolymerase that can perform rolling circle replication in the presenceof a strand displacement factor is suitable for use in the disclosedmethod, even if the DNA polymerase does not perform rolling circlereplication in the absence of such a factor. Strand displacement factorsuseful in RCA include BMRF1 polymerase accessory subunit (Tsurumi etal., J. Virology 67(12):7648-7653 (1993)), adenovirus DNA-bindingprotein (Zijderveld and van der Vliet, J. Virology 68(2):1158-1164(1994)), herpes simplex viral protein ICP8 (Boehmer and Lehman, J.Virology 67(2):711-715 (1993); Skaliter and Lehman, Proc. Natl. Acad.Sci. USA 91(22):10665-10669 (1994)), single-stranded DNA bindingproteins (SSB; Rigler and Romano, J. Biol. Chem. 270:8910-8919 (1995)),and calf thymus helicase (Siegel et al., J. Biol. Chem. 267:13629-13635(1992)).

The ability of a polymerase to carry out rolling circle replication canbe determined by using the polymerase in a rolling circle replicationassay such as those described in Fire and Xu, Proc. Natl. Acad. Sci. USA92:4641-4645 (1995) and in Example 1.

Another type of DNA polymerase can be used if a gap-filling synthesisstep is used, such as in gap-filling LM-RCA (see Example 3). When usinga DNA polymerase to fill gaps, strand displacement by the DNA polymeraseis undesirable. Such DNA polymerases are referred to herein asgap-filling DNA polymerases. Unless otherwise indicated, a DNApolymerase referred to herein without specifying it as a rolling circleDNA polymerase or a gap-filling DNA polymerase, is understood to be arolling circle DNA polymerase and not a gap-filling DNA polymerase.Preferred gap-filling DNA polymerases are T7 DNA polymerase (Studier etal., Methods Enzymol. 185:60-89 (1990)), DEEP VENT® DNA polymerase (NewEngland Biolabs, Beverly, Mass.), modified T7 DNA polymerase (Tabor andRichardson, J. Biol. Chem. 262:15330-15333 (1987); Tabor and Richardson,J. Biol. Chem. 264:6447-6458 (1989); Sequenase™ (U.S. Biochemicals)),and T4 DNA polymerase (Kunkel et al., Methods Enzymol. 154:367-382(1987)). An especially preferred type of gap-filling DNA polymerase isthe Thermus flavus DNA polymerase (MBR, Milwaukee, Wis.). The mostpreferred gap-filling DNA polymerase is the Stoffel fragment of Taq DNApolymerase (Lawyer et al., PCR Methods Appl. 2(4):275-287 (1993), Kinget al., J. Biol. Chem. 269(18):13061-13064 (1994)).

The ability of a polymerase to fill gaps can be determined by performinggap-filling LM-RCA. Gap-filling LM-RCA is performed with an open circleprobe that forms a gap space when hybridized to the target sequence.Ligation can only occur when the gap space is filled by the DNApolymerase. If gap-filling occurs, TS-DNA can be detected, otherwise itcan be concluded that the DNA polymerase, or the reaction conditions, isnot useful as a gap-filling DNA polymerase.

T. RNA Polymerases

Any RNA polymerase which can carry out transcription in vitro and forwhich promoter sequences have been identified can be used in thedisclosed rolling circle transcription method. Stable RNA polymeraseswithout complex requirements are preferred. Most preferred are T7 RNApolymerase (Davanloo et al., Proc. Natl. Acad. Sci. USA 81:2035-2039(1984)) and SP6 RNA polymerase (Butler and Chamberlin, J. Biol. Chem.257:5772-5778 (1982)) which are highly specific for particular promotersequences (Schenborn and Meirendorf, Nucleic Acids Research 13:6223-6236(1985)). Other RNA polymerases with this characteristic are alsopreferred. Because promoter sequences are generally recognized byspecific RNA polymerases, the OCP or ATC should contain a promotersequence recognized by the RNA polymerase that is used. Numerouspromoter sequences are known and any suitable RNA polymerase having anidentified promoter sequence can be used. Promoter sequences for RNApolymerases can be identified using established techniques.

The materials described above can be packaged together in any suitablecombination as a kit useful for performing the disclosed method.

II. Method

The disclosed rolling circle amplification (RCA) method involvesreplication of circular single-stranded DNA molecules. In RCA, a rollingcircle replication primer hybridizes to circular OCP or ATC moleculesfollowed by rolling circle replication of the OCP or ATC molecules usinga strand-displacing DNA polymerase. Amplification takes place duringrolling circle replication in a single reaction cycle. Rolling circlereplication results in large DNA molecules containing tandem repeats ofthe OCP or ATC sequence. This DNA molecule is referred to as a tandemsequence DNA (TS-DNA). Rolling circle amplification is also referred toherein as unimolecular segment amplification (USA). The termunimolecular segment amplification is generally used herein to emphasisthe amplification of individual segments of nucleic acid, such as atarget sequence, that are of interest.

A preferred embodiment, ligation-mediated rolling circle amplification(LM-RCA) method involves a ligation operation prior to replication. Inthe ligation operation, an OCP hybridizes to its cognate target nucleicacid sequence, if present, followed by ligation of the ends of thehybridized OCP to form a covalently closed, single-stranded OCP. Afterligation, a rolling circle replication primer hybridizes to OCPmolecules followed by rolling circle replication of the circular OCPmolecules using a strand-displacing DNA polymerase. Generally, LM-RCAcomprises

(a) mixing an open circle probe (OCP) with a target sample, resulting inan OCP-target sample mixture, and incubating the OCP-target samplemixture under conditions promoting hybridization between the open circleprobe and a target sequence,

(b) mixing ligase with the OCP-target sample mixture, resulting in aligation mixture, and incubating the ligation mixture under conditionspromoting ligation of the open circle probe to form an amplificationtarget circle (ATC),

(c) mixing a rolling circle replication primer (RCRP) with the ligationmixture, resulting in a primer-ATC mixture, and incubating theprimer-ATC mixture under conditions that promote hybridization betweenthe amplification target circle and the rolling circle replicationprimer,

(d) mixing DNA polymerase with the primer-ATC mixture, resulting in apolymerase-ATC mixture, and incubating the polymerase-ATC mixture underconditions promoting replication of the amplification target circle,where replication of the amplification target circle results information of tandem sequence DNA (TS-DNA).

The open circle probe is a single-stranded, linear DNA moleculecomprising, from 5' end to 3' end, a 5' phosphate group, a right targetprobe portion, a primer complement portion, a spacer region, a lefttarget probe portion, and a 3' hydroxyl group, wherein the left targetprobe portion is complementary to the 5' region of a target sequence andthe right target probe portion is complementary to the 3' region of thetarget sequence.

The left and right target probe portions hybridize to the two ends ofthe target nucleic acid sequence, with or without a central gap to befilled by one or more gap oligonucleotides. Generally, LM-RCA using gapoligonucleotides can be performed by, in an LM-RCA reaction, (1) using atarget sequence with a central region located between a 5' region and a3' region, and an OCP where neither the left target probe portion of theopen circle probe nor the right target probe portion of the open circleprobe is complementary to the central region of the target sequence, and(2) mixing one or more gap oligonucleotides with the target sample, suchthat the OCP-target sample mixture contains the open circle probe, theone or more gap oligonucleotides, and the target sample, where each gapoligonucleotide consists of a single-stranded, linear DNA moleculecomprising a 5' phosphate group and a 3' hydroxyl group, where each gapoligonucleotide is complementary all or a portion of the central regionof the target sequence.

A. The Ligation Operation

An open circle probe, optionally in the presence of one or more gapoligonucleotides, is incubated with a sample containing DNA, RNA, orboth, under suitable hybridization conditions, and then ligated to forma covalently closed circle. The ligated open circle probe is a form ofamplification target circle. This operation is similar to ligation ofpadlock probes described by Nilsson et al., Science, 265:2085-2088(1994). The ligation operation allows subsequent amplification to bedependent on the presence of a target sequence. Suitable ligases for theligation operation are described above. Ligation conditions aregenerally known. Most ligases require Mg⁺⁺. There are two main types ofligases, those that are ATP-dependent and those that are NAD-dependent.ATP or NAD, depending on the type of ligase, should be present duringligation.

The ligase and ligation conditions can be optimized to limit thefrequency of ligation of single-stranded termini. Such ligation eventsdo not depend on the presence of a target sequence. In the case ofAMPLIGASE® -catalyzed ligation, which takes place at 60° C., it isestimated that no more than 1 in 1,000,000 molecules withsingle-stranded DNA termini will be ligated. This is based on the levelof non-specific amplification seen with this ligase in the ligase chainreaction. Any higher nonspecific ligation frequency would causeenormously high background amplification in the ligase chain reaction.Using this estimate, an approximate frequency for the generation ofnon-specifically ligated open circles with a correctly placed gapoligonucleotide in at the ligation junction can be calculated. Since twoligation events are involved, the frequency of such events usingAMPLIGASE® should be the square of 1 in 1,000,000, or 1 in 1×10¹². Thenumber of probes used in a typical ligation reaction of 50 μl is 2×10¹².Thus, the number of non-specifically ligated circles containing acorrect gap oligonucleotide would be expected to be about 2 perreaction.

When RNA is to be detected, it is preferred that a reverse transcriptionoperation be performed to make a DNA target sequence. An example of theuse of such an operation is described in Example 4. Alternatively, anRNA target sequence can be detected directly by using a ligase that canperform ligation on a DNA:RNA hybrid substrate. A preferred ligase forthis is T4 DNA ligase.

B. The Replication Operation

The circular open circle probes formed by specific ligation andamplification target circles serve as substrates for a rolling circlereplication. This reaction requires the addition of two reagents: (a) arolling circle replication primer, which is complementary to the primercomplement portion of the OCP or ATC, and (b) a rolling circle DNApolymerase. The DNA polymerase catalyzes primer extension and stranddisplacement in a processive rolling circle polymerization reaction thatproceeds as long as desired, generating a molecule of up to 100,000nucleotides or larger that contains up to approximately 1000 tandemcopies of a sequence complementary to the amplification target circle oropen circle probe (FIG. 4). This tandem sequence DNA (TS-DNA) consistsof, in the case of OCPs, alternating target sequence and spacersequence. Note that the spacer sequence of the TS-DNA is the complementof the sequence between the left target probe and the right target probein the original open circle probe. A preferred rolling circle DNApolymerase is the DNA polymerase of the bacteriophage φ29.

During rolling circle replication one may additionally includeradioactive, or modified nucleotides such as bromodeoxyuridinetriphosphate, in order to label the DNA generated in the reaction.Alternatively, one may include suitable precursors that provide abinding moiety such as biotinylated nucleotides (Langer et al. (1981)).

Rolling circle amplification can be engineered to produce TS-DNA ofdifferent lengths in an assay involving multiple ligated OCPs or ATCs.This can be useful for extending the number of different targets thatcan be detected in a single assay. TS-DNA of different lengths can beproduced in several ways. In one embodiment, the base composition of thespacer region of different classes of OCP or ATC can be designed to berich in a particular nucleotide. Then a small amount of the dideoxynucleotide complementary to the enriched nucleotide can be included inthe rolling circle amplification reaction. After some amplification, thedideoxy nucleotides will terminate extension of the TS-DNA product ofthe class of OCP or ATC enriched for the complementary nucleotide. OtherOCPs or ATCs will be less likely to be terminated, since they are notenriched for the complementary nucleotide, and will produce longerTS-DNA products, on average.

In another embodiment, two different classes of OCP or ATC can bedesigned with different primer complement portions. These differentprimer complement portions are designed to be complementary to adifferent rolling circle replication primer. Then the two differentrolling circle replication primers are used together in a single rollingcircle amplification reaction, but at significantly differentconcentrations. The primer at high concentration immediately primesrolling circle replication due to favorable kinetics, while the primerat lower concentration is delayed in priming due to unfavorablekinetics. Thus, the TS-DNA product of the class of OCP or ATC designedfor the primer at high concentration will be longer than the TS-DNAproduct of the class of OCP or ATC designed for the primer at lowerconcentration since it will have been replicated for a longer period oftime. As another option, one of the rolling circle replication primerscan be a caged oligonucleotide. In this case, the two rolling circlereplication primers can be at similar concentrations. The caged rollingcircle replication primer will not support rolling circle replicationuntil the cage structure is removed. Thus, the first, uncaged rollingcircle replication primer begins amplification of its cognateamplification target circle(s) when the replication operation begins,the second, caged rolling circle replication primer begins amplificationof its cognate amplification target circle(s) only after removal of thecage. The amount of TS-DNA produced from each rolling circle replicationprimer will differ proportionate to the different effective times ofreplication. Thus, the amount of TS-DNA made using each type of rollingcircle replication primer can be controlled using a caged primer. Theuse of such a caged primer has the advantage that the caged primer canbe provided at a sufficient concentration to efficiently initiaterolling circle replication as soon as it is uncaged (rather than at alower concentration).

C. Modifications And Additional Operations

1. Detection of Amplification Products

Current detection technology makes a second cycle of RCA unnecessary inmany cases. Thus, one may proceed to detect the products of the firstcycle of RCA directly. Detection may be accomplished by primary labelingor by secondary labeling, as described below.

(a) Primary Labeling

Primary labeling consists of incorporating labeled moieties, such asfluorescent nucleotides, biotinylated nucleotides,digoxygenin-containing nucleotides, or bromodeoxyuridine, during rollingcircle replication in RCA, or during transcription in RCT. For example,one may incorporate cyanine dye UTP analogs (Yu et al. (1994)) at afrequency of 4 analogs for every 100 nucleotides. A preferred method fordetecting nucleic acid amplified in situ is to label the DNA duringamplification with BrdUrd, followed by binding of the incorporated BUDRwith a biotinylated anti-BUDR antibody (Zymed Labs, San Francisco,Calif.), followed by binding of the biotin moieties withStreptavidin-Peroxidase (Life Sciences, Inc.), and finally developmentof fluorescence with Fluorescein-tyramide (DuPont de Nemours & Co.,Medical Products Dept.).

(b) Secondary Labeling with Detection Probes

Secondary labeling consists of using suitable molecular probes, referredto as detection probes, to detect the amplified DNA or RNA. For example,an open circle may be designed to contain several repeats of a knownarbitrary sequence, referred to as detection tags. A secondaryhybridization step can be used to bind detection probes to thesedetection tags (FIG. 7). The detection probes may be labeled asdescribed above with, for example, an enzyme, fluorescent moieties, orradioactive isotopes. By using three detection tags per open circleprobe, and four fluorescent moieties per each detection probe, one mayobtain a total of twelve fluorescent signals for every open circle proberepeat in the TS-DNA, yielding a total of 12,000 fluorescent moietiesfor every ligated open circle probe that is amplified by RCA.

(c) Multiplexing and Hybridization Array Detection

RCA is easily multiplexed by using sets of different open circle probes,each set carrying different target probe sequences designed for bindingto unique targets. Note that although the target probe sequencesdesigned for each target are different, the primer complement portionmay remain unchanged, and thus the primer for rolling circle replicationcan remain the same for all targets. Only those open circle probes thatare able to find their targets will give rise to TS-DNA. The TS-DNAmolecules generated by RCA are of high molecular weight and lowcomplexity; the complexity being the length of the open circle probe.There are two alternatives for capturing a given TS-DNA to a fixedposition in a solid-state detector. One is to include within the spacerregion of the open circle probes a unique address tag sequence for eachunique open circle probe. TS-DNA generated from a given open circleprobe will then contain sequences corresponding to a specific addresstag sequence. A second and preferred alternative is to use the targetsequence present on the TS-DNA as the address tag.

(d) Combinatorial Multicolor Coding

A preferred form of multiplex detection involves the use of acombination of labels that either fluoresce at different wavelengths orare colored differently. One of the advantages of fluorescence for thedetection of hybridization probes is that several targets can bevisualized simultaneously in the same sample. Using a combinatorialstrategy, many more targets can be discriminated than the number ofspectrally resolvable fluorophores. Combinatorial labeling provides thesimplest way to label probes in a multiplex fashion since a probe fluoris either completely absent (-) or present in unit amounts (+); imageanalysis is thus more amenable to automaton, and a number ofexperimental artifacts, such as differential photobleaching of thefluors and the effects of changing excitation source power spectrum, areavoided.

The combinations of labels establish a code for identifying differentdetection probes and, by extension, different target molecules to whichthose detection probes are associated with. This labeling scheme isreferred to as Combinatorial Multicolor Coding (CMC). Such coding isdescribed by Speicher et al., Nature Genetics 12:368-375 (1996). Anynumber of labels, which when combined can be separately detected, can beused for combinatorial multicolor coding. It is preferred that 2, 3, 4,5, or 6 labels be used in combination. It is most preferred that 6labels be used. The number of labels used establishes the number ofunique label combinations that can be formed according to the formula2^(N) -1, where N is the number of labels. According to this formula, 2labels forms three label combinations, 3 labels forms seven labelcombinations, 4 labels forms 15 label combinations, 5 labels form 31label combinations, and 6 labels forms 63 label combinations.

For combinatorial multicolor coding, a group of different detectionprobes are used as a set. Each type of detection probe in the set islabeled with a specific and unique combination of fluorescent labels.For those detection probes assigned multiple labels, the labeling can beaccomplished by labeling each detection probe molecule with all of therequired labels. Alternatively, pools of detection probes of a giventype can each be labeled with one of the required labels. By combiningthe pools, the detection probes will, as a group, contain thecombination of labels required for that type of detection probe. Thiscan be illustrated with a simple example. Starting with seven differenttypes of detection probe, each complementary to a different detectiontag and designated 1 through 7, unique identification requires threedifferent labels used in seven combinations. Assigning the combinationsarbitrarily, one coding scheme is:

    ______________________________________                                        Detection probe                                                                         1        2     3     4   5     6   7                                ______________________________________                                        label A   +                    +   +         +                                  label B  +  +  + +                                                            label C   +  + + +                                                          ______________________________________                                    

As can be seen, detection probe 7 must be labeled with three differentlabels, A, B, and C. This can be accomplished by labels A, B, and C toeach individual detection probe 7 molecule. This is the first optiondescribed above. Alternatively, three pools of detection probe 7 can beseparately labeled, one pool with label A, one pool with label B, andone pool with label C. In each pool, individual detection molecules arelabeled with a single type of label. Mixing the pools results in asolution of detection probe 7 that collectively contains all threelabels as required. Labeling of detection probes requiring differentnumbers of probes can be accomplished in a similar fashion.

Of course, the two types of labeling schemes described above can becombined, resulting in detection probe molecules with multiple labelscombined with detection probe molecules of the same type multiplylabeled with different labels. This can be illustrated using the exampleabove. Two pools of detection probe type 7 can be separately labeled,one pool with both labels A and B, and one pool with only label C.Mixing the pools results in a solution of detection probe 7 thatcollectively contains all three labels as required. Combinatorialmulticolor coding is further illustrated in Examples 7 and 8.

Where each detection probe is labeled with a single label, labelcombinations can also be generated by using OCPs or ATCs with codedcombinations of detection tags complementary to the different detectionprobes. In this scheme, the OCPs or ATCs will contain a combination ofdetection tags representing the combination of labels required for aspecific label code. Using the example above, a set of seven OCPs orATCs, designated 1 though 7, would contain one, two, or three detectiontags, chosen from a set of three detection tag sequences designated dtA,dtB, and dtC. Each detection tag sequence corresponds to one of thelabels, A, B, or C, with each label coupled to one of three detectionprobes, designated dpA, dpB, or dpC, respectively. An example of theresulting coding scheme would be:

    ______________________________________                                        OCP or ATC                                                                              1       2     3     4   5     6   7                                 ______________________________________                                        dtA       +                   +   +         +                                   dtB  +  +  + +                                                                dtC   +  + + +                                                              ______________________________________                                    

Hybridization could be performed with a pool of all the differentlabeled detection probes, dpA, dpB, and dpC. The result would be thatTS-DNA generated from OCP 7 would hybridize to all three detectionprobes, thus labeling the TS-DNA with all three labels. In contrast,TS-DNA generated from OCP 4, for example, would hybridize only todetection probes dpA and dpB, thus labeling the OCP 4-derived TS-DNAwith only labels A and B. This method of coding and detection ispreferred. Use of this coding scheme is illustrated in Examples 7 and 8.

As described above, rolling circle amplification can be engineered toproduce TS-DNA of different lengths in an assay involving multipleligated OCPs or ATCs. The resulting TS-DNA of different length can bedistinguished simply on the basis of the size of the detection signalthey generate. Thus, the same set of detection probes could be used todistinguish two different sets of generated TS-DNA. In this scheme, twodifferent TS-DNAs, each of a different size but assigned the same colorcode, would be distinguished by the size of the signal produced by thehybridized detection probes. In this way, a total of 126 differenttargets can be distinguished on a single solid-state sample using a codewith 63 combinations, since the signals will come in two flavors, lowamplitude and high amplitude. Thus one could, for example, use the lowamplitude signal set of 63 probes for detection of an oncogenemutations, and the high amplitude signal set of 63 probes for thedetection of a tumor suppressor p53 mutations.

Speicher et al. describes a set of fluors and corresponding opticalfilters spaced across the spectral interval 350-770 nm that give a highdegree of discrimination between all possible fluor pairs. This fluorset, which is preferred for combinatorial multicolor coding, consists of4'-6-diamidino-2-phenylinodole (DAPI), fluorescein (FITC), and thecyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. Any subset of thispreferred set can also be used where fewer combinations are required.The absorption and emission maxima, respectively, for these fluors are:DAPI (350 nm; 456 nm), FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm),Cy3.5 (581 nm; 588 nm), Cy5 (652 nm; 672 nm), Cy5.5 (682 nm; 703 nm) andCy7 (755 nm; 778 nm). The excitation and emission spectra, extinctioncoefficients and quantum yield of these fluors are described by Ernst etal., Cytometry 10:3-10 (1989), Mujumdar et al., Cytometry 10:11-19(1989), Yu, Nucleic Acids Res. 22:3226-3232 (1994), and Waggoner, Meth.Enzymology 246:362-373 (1995). These fluors can all be excited with a75W Xenon arc.

To attain selectivity, filters with bandwidths in the range of 5 to 16nm are preferred. To increase signal discrimination, the fluors can beboth excited and detected at wavelengths far from their spectral maxima.Emission bandwidths can be made as wide as possible. For low-noisedetectors, such as cooled CCD cameras, restricting the excitationbandwidth has little effect on attainable signal to noise ratios. A listof preferred filters for use with the preferred fluor set is listed inTable 1 of Speicher et al. It is important to prevent infra-red lightemitted by the arc lamp from reaching the detector; CCD chips areextremely sensitive in this region. For this purpose, appropriate IRblocking filters can be inserted in the image path immediately in frontof the CCD window to minimize loss of image quality. Image analysissoftware can then be used to count and analyze the spectral signaturesof fluorescent dots.

Discrimination of individual signals in combinatorial multicolor codingcan be enhanced by collapsing TS-DNA generated during amplification. Asdescribed above, this is preferably accomplished using collapsingdetection probes, biotin-antibody conjugates, or a combination of both.A collapsed TS-DNA can occupy a space of no more than 0.3 microns indiameter. Based on this, it is expected that up to a million discretesignals can be detected in a 2.5 mm sample dot. Such discrimination alsoresults in a large dynamic range for quantitative signal detection. Forexample, where two separate signals are detected in the same sample dot,a ratio of the two signals up to 1:500,000 can be detected. Thus, therelative numbers of different types of signals (such as multicolorcodes) can be determined over a wide range. This is expected to allowdetermination of, for example, whether a particular target sequence ishomozygous or heterozygous in a genomic DNA sample, whether a targetsequence was inherited or represents a somatic mutation, and the geneticheterogeneity of a genomic DNA sample, such as a tumor sample. In thefirst case, a homozygous target sequence would produce twice the numberof signals of a heterozygous target sequence. In the second case, aninherited target sequence would produce a number of signals equivalentto a homozygous or heterozygous signal (that is, a large number ofsignals), while a somatic mutation would produce a smaller number ofsignals depending on the source of the sample. In the third case, therelative number of cells, from which a sample is derived, that haveparticular target sequences can be determined. The more cells in thesample with a particular target sequence, the larger the signal.

(e) Detecting Groups of Target Sequences

Multiplex RCA assays are particularly useful for detecting mutations ingenes where numerous distinct mutations are associated with certaindiseases or where mutations in multiple genes are involved. For example,although the gene responsible for Huntington's chorea has beenidentified, a wide range of mutations in different parts of the geneoccur among affected individuals. The result is that no single test hasbeen devised to detect whether an individual has one or more of the manyHuntington's mutations. A single LM-RCA assay can be used to detect thepresence of one or more members of a group of any number of targetsequences. This can be accomplished, for example, by designing an opencircle probe (and associated gap oligonucleotides, if desired) for eachtarget sequence in the group, where the target probe portions of eachopen circle probe are different but the sequence of the primer portionsand the sequence of the detection tag portions of all the open circleprobes are the same. All of the open circle probes are placed in thesame OCP-target sample mixture, and the same primer and detection probeare used to amplify and detect TS-DNA. If any of the target sequencesare present in the target sample, the OCP for that target will beligated into a circle and the circle will be amplified to form TS-DNA.Since the detection tags on TS-DNA resulting from amplification of anyof the OCPs are the same, TS-DNA resulting from ligation of any of theOCPs will be detected in that assay. Detection indicates that at leastone member of the target sequence group is present in the target sample.This allows detection of a trait associated with multiple targetsequences in a single tube or well.

If a positive result is found, the specific target sequence involved canbe identified by using a multiplex assay. This can be facilitated byincluding an additional, different detection tag in each of the OCPs ofthe group. In this way, TS-DNA generated from each different OCP,representing each different target sequence, can be individuallydetected. It is convenient that such multiple assays need be performedonly when an initial positive result is found.

The above scheme can also be used with arbitrarily chosen groups oftarget sequences in order to screen for a large number of targetsequences without having to perform an equally large number of assays.Initial assays can be performed as described above, each using adifferent group of OCPs designed to hybridize to a different group oftarget sequences. Additional assays to determine which target sequenceis present can then be performed on only those groups that produceTS-DNA. Such group assays can be further nested if desired.

(f) In Situ Detection Using RCA

In situ hybridization, and its most powerful implementation, known asfluorescent in situ hybridization (FISH), is of fundamental importancein cytogenetics. RCA can be adapted for use in FISH, as follows.

Open circle probes are ligated on targets on microscope slides, andincubated in situ with fluorescent precursors during rolling circlereplication. The rolling circle DNA polymerase displaces the ligatedopen circle probe from the position where it was originally hybridized.However, the circle will remain topologically trapped on the chromosomeunless the DNA is nicked (Nilsson et al. (1994)). The presence ofresidual chromatin may slow diffusion of the circle along thechromosome. Alternatively, fixation methods may be modified to minimizethis diffusional effect. This diffusion has an equal probability ofoccurring in either of two directions along the chromosome, and hencenet diffusional displacement may be relatively small during a 10 minuteincubation. During this time rolling circle replication should generatea linear molecule of approximately 25,000 nucleotides containingapproximately 2,500 bromodeoxyuridine moieties, which can be detectedwith a biotinylated anti-BUDR IgG (Zymed Labs, Inc.) andfluorescein-labeled avidin. This level of incorporation shouldfacilitate recording of the image using a microscope-based CCD system.Diffusion may also be limited because the TS-DNA should be able tohybridize with the complement of the target strand.

A preferred method of in situ detection is Reporter Binding AgentUnimolecular Rolling Amplification (RBAURA), which is described below.In RBAURA, a reporter binding agent is used where the oligonucleotideportion serves as a rolling circle replication primer. Once the reporterbinding agent is associated with a target molecule, an amplificationtarget circle is hybridized to the rolling circle replication primersequence of the reporter binding agent followed by amplification of theATC by RCA. The resulting TS-DNA has the rolling circle replicationprimer sequence of the reporter binding agent at one end, thus anchoringthe TS-DNA to the site of the target molecule. Peptide Nucleic AcidProbe Unimolecular Rolling Amplification (PNAPURA) and Locked AntibodyUnimolecular Rolling Amplification (LAURA), described below, arepreferred forms of RBAURA.

Localization of the TS-DNA for in situ detection can also be enhanced bycollapsing the TS-DNA using collapsing detection probes, biotin-antibodyconjugates, or both, as described above. Multiplexed in situ detectioncan be carried out as follows: Rolling circle replication is carried outusing unlabeled nucleotides. The different TS-DNAs are then detectedusing standard multi-color FISH with detection probes specific for eachunique target sequence or each unique detection tag in the TS-DNA.Alternatively, and preferably, combinatorial multicolor coding, asdescribed above, can be used for multiplex in situ detection.

(g) Enzyme-linked Detection

Amplified nucleic acid labeled by incorporation of labeled nucleotidescan be detected with established enzyme-linked detection systems. Forexample, amplified nucleic acid labeled by incorporation ofbiotin-16-UTP (Boehringher Mannheim) can be detected as follows. Thenucleic acid is immobilized on a solid glass surface by hybridizationwith a complementary DNA oligonucleotide (address probe) complementaryto the target sequence (or its complement) present in the amplifiednucleic acid. After hybridization, the glass slide is washed andcontacted with alkaline phosphatase-streptavidin conjugate (Tropix,Inc., Bedford, Mass.). This enzyme-streptavidin conjugate binds to thebiotin moieties on the amplified nucleic acid. The slide is again washedto remove excess enzyme conjugate and the chemiluminescent substrateCSPD (Tropix, Inc.) is added and covered with a glass cover slip. Theslide can then be imaged in a Biorad Fluorimager.

(h) Collapse of Nucleic Acids

As described above, TS-DNA or TS-RNA, which are produced as extendednucleic acid molecules, can be collapsed into a compact structure. Itshould also be understood that the same collapsing procedure can beperformed on any extended nucleic acid molecule. For example, genomicDNA, PCR products, viral RNA or DNA, and cDNA samples can all becollapsed into compact structures using the disclosed collapsingprocedure. It is preferred that the nucleic acid to be collapsed isimmobilized on a substrate. A preferred means of collapsing nucleicacids is by hybridizing one or more collapsing probes with the nucleicacid to be collapsed. Collapsing probes are oligonucleotides having aplurality of portions each complementary to sequences in the nucleicacid to be collapsed. These portions are referred to as complementaryportions of the collapsing probe, where each complementary portion iscomplementary to a sequence in the nucleic acid to be collapsed. Thesequences in the nucleic acid to be collapsed are referred to ascollapsing target sequences. The complementary portion of a collapsingprobe can be any length that supports specific and stable hybridizationbetween the collapsing probe and the collapsing target sequence. Forthis purpose, a length of 10 to 35 nucleotides is preferred, with acomplementary portion of a collapsing probe 16 to 20 nucleotides longbeing most preferred. It is preferred that at least two of thecomplementary portions of a collapsing probe be complementary tocollapsing target sequences which are separated on the nucleic acid tobe collapsed or to collapsing target sequences present in separatenucleic acid molecules. This allows each detection probe to hybridize toat least two separate collapsing target sequences in the nucleic acidsample. In this way, the collapsing probe forms a bridge betweendifferent parts of the nucleic acid to be collapsed. The combined actionof numerous collapsing probes hybridizing to the nucleic acid will be toform a collapsed network of cross-linked nucleic acid. Collapsed nucleicacid occupies a much smaller volume than free, extended nucleic acid,and includes whatever detection probe or detection label hybridized tothe nucleic acid. This result is a compact and discrete nucleic acidstructure which can be more easily detected than extended nucleic acid.Collapsing nucleic acids is useful both for in situ hybridizationapplications and for multiplex detection because it allows detectablesignals to be spatially separate even when closely packed. Collapsingnucleic acids is especially preferred for use with combinatorialmulticolor coding.

Collapsing probes can also contain any of the detection labels describedabove. This allows detection of the collapsed nucleic acid in caseswhere separate detection probes or other means of detecting the nucleicacid are not employed. Preferred labels are biotin and fluorescentmolecules. A particularly preferred detection probe is a molecularbeacon. Molecular beacons are detection probes labeled with fluorescentmoieties where the fluorescent moieties fluoresce only when thedetection probe is hybridized. The use of such probes eliminates theneed for removal of unhybridized probes prior to label detection becausethe unhybridized detection probes will not produce a signal. This isespecially useful in multiplex assays.

TS-DNA collapse can also be accomplished through the use ofligand/ligand binding pairs (such as biotin and avidin) orhapten/antibody pairs. As described in Example 6, a nucleotide analog,BUDR, can be incorporated into TS-DNA during rolling circle replication.When biotinylated antibodies specific for BUDR and avidin are added, across-linked network of TS-DNA forms, bridged by avidin-biotin-antibodyconjugates, and the TS-DNA collapses into a compact structure.Biotin-derivatized nucleic acid can be formed in many of the commonnucleic acid replication operations such as cDNA synthesis, PCR, andother nucleic acid amplification techniques. In most cases, biotin canbe incorporated into the synthesized nucleic acid by eitherincorporation of biotin-derivatized nucleotides or through the use ofbiotin-derivatized primers. Collapsing probes and biotin-mediatedcollapse can also be used together to collapse nucleic acids.

2. Nested LM-RCA

After RCA, a round of LM-RCA can be performed on the TS-DNA produced inthe first RCA. This new round of LM-RCA is performed with a new opencircle probe, referred to as a secondary open circle probe, havingtarget probe portions complementary to a target sequence in the TS-DNAproduced in the first round. When such new rounds of LM-RCA areperformed, the amplification is referred to herein as nested LM-RCA.Nested LM-RCA is particularly useful for in situ hybridizationapplications of LM-RCA. Preferably, the target probe portions of thesecondary OCP are complementary to a secondary target sequence in thespacer sequences of the TS-DNA produced in the first RCA. The complementof this secondary target sequence is present in the spacer portion ofthe OCP or ATC used in the first RCA. After mixing the secondary OCPwith the TS-DNA, ligation and rolling circle amplification proceed as inLM-RCA. Each ligated secondary OCP generates a new TS-DNA. By having,for example, two secondary target sequence portions in the first roundOCP, the new round of LM-RCA will yield two secondary TS-DNA moleculesfor every OCP or ATC repeat in the TS-DNA produced in the first RCA.Thus, the amplification yield of nested LM-RCA is about 2000-fold. Theoverall amplification using two cycles of RCA is thus1000×2000=2,000,000. Nested LM-RCA can follow any DNA replication ortranscription operation described herein, such as RCA, LM-RCA, secondaryDNA strand displacement, strand displacement cascade amplification, ortranscription.

Generally, nested LM-RCA involves, following a first RCA,

(a) mixing a secondary open circle probe with the polymerase mixture,resulting in an OCP-TS mixture, and incubating the OCP-TS mixture underconditions promoting hybridization between the secondary open circleprobe and the tandem sequence DNA,

(b) mixing ligase with the OCP-TS mixture, resulting in a secondaryligation mixture, and incubating the secondary ligation mixture underconditions promoting ligation of the secondary open circle probe to forma secondary amplification target circle,

(c) mixing a rolling circle replication primer with the secondaryligation mixture, resulting in a secondary primer-ATC mixture, andincubating the secondary primer-ATC mixture under conditions thatpromote hybridization between the secondary amplification target circleand rolling circle replication primer,

(d) mixing DNA polymerase with the secondary primer-ATC mixture,resulting in a secondary polymerase-ATC mixture, and incubating thesecondary polymerase-ATC mixture under conditions promoting replicationof the secondary amplification target circle, where replication of thesecondary amplification target circle results in formation of nestedtandem sequence DNA.

An exonuclease digestion step can be added prior to performing thenested LM-RCA. This is especially useful when the target probe portionsof the secondary open circle probe are the same as those in the firstopen circle probe. Any OCP which has been ligated will not be digestedsince ligated OCPs have no free end. A preferred way to digest OCPs thathave hybridized to TS-DNA during the first round of LM-RCA is to use aspecial rolling circle replication primer containing at least about fourphosphorothioate linkages between the nucleotides at the 5' end. Then,following rolling circle replication, the reaction mixture is subjectedto exonuclease digestion. By using a 5' exonuclease unable to cleavethese phosphorothioate linkages, only the OCPs hybridized to TS-DNA willbe digested, not the TS-DNA. The TS-DNA generated during the first cycleof amplification will not be digested by the exonuclease because it isprotected by the phosphorothioate linkages at the 5' end. A preferredexonuclease for this purpose is the T7 gene 6 exonuclease. The T7 gene 6exonuclease can be inactivated prior to adding the secondary open circleprobe by heating to 90° C. for 10 minutes.

By using an exonuclease digestion, nested LM-RCA can be performed usingthe same target sequence used in a first round of LM-RCA. This can bedone, for example, generally as follows. After the first round ofLM-RCA, the unligated open circle probes and gap oligonucleotideshybridized to TS-DNA are digested with T7 gene 6 exonuclease. Theexonuclease is inactivated by heating for 10 minutes at 90° C. Then asecond open circle probe is added. In this scheme, the second opencircle probe has target probe portions complementary to the sameoriginal target sequence, but which contain a different (arbitrary)spacer region sequence. A second round of LM-RCA is then performed. Inthis second round, the target of the second open circle probes comprisesthe repeated target sequences contained in the TS-DNA generated by thefirst cycle. This procedure has the advantage of preserving the originaltarget sequence in the amplified DNA obtained after nested LM-RCA.

Nested LM-RCA can also be performed on ligated OCPs or ATCs that havenot been amplified. In this case, LM-RCA can be carried out using eitherATCs or target-dependent ligated OCPs. This is especially useful for insitu detection. For in situ detection, the first, unamplified OCP, whichis topologically locked to its target sequence, can be subjected tonested LM-RCA. By not amplifying the first OCP, it can remain hybridizedto the target sequence while LM-RCA amplifies a secondary OCPtopologically locked to the first OCP. This is illustrated in FIG. 12.

3. Secondary DNA strand displacement and Strand Displacement CascadeAmplification

Secondary DNA strand displacement is another way to amplify TS-DNA.Secondary DNA strand displacement is accomplished by hybridizingsecondary DNA strand displacement primers to TS-DNA and allowing a DNApolymerase to synthesize DNA from these primed sites (FIG. 11). Since acomplement of the secondary DNA strand displacement primer occurs ineach repeat of the TS-DNA, secondary DNA strand displacement can resultin a level of amplification similar to or larger than that obtained inRCT. The product of secondary DNA strand displacement is referred to assecondary tandem sequence DNA or TS-DNA-2.

Secondary DNA strand displacement can be accomplished by performing RCAto produce TS-DNA in a polymerase-ATC mixture, and then mixing secondaryDNA strand displacement primer with the polymerase-ATC mixture,resulting in a secondary DNA strand displacement mixture, and incubatingthe secondary DNA strand displacement mixture under conditions promotingreplication of the tandem sequence DNA. The secondary DNA stranddisplacement primer is complementary to a part of the OCP or ATC used togenerated TS-DNA as described earlier. It is preferred that thesecondary DNA strand displacement primer is not complementary to therolling circle replication primer, or to a tertiary DNA stranddisplacement primer, if used.

Secondary DNA strand displacement can also be carried out simultaneouslywith rolling circle replication. This is accomplished by mixingsecondary DNA strand displacement primer with the polymerase-ATC mixtureprior to incubating the mixture for rolling circle replication. Forsimultaneous rolling circle replication and secondary DNA stranddisplacement, it is preferred that the rolling circle DNA polymerase beused for both replications. This allows optimum conditions to be usedand results in displacement of other strands being synthesizeddownstream as shown in FIG. 11B. Secondary DNA strand displacement canfollow any DNA replication operation disclosed herein, such as RCA,LM-RCA or nested LM-RCA.

To optimize the efficiency of secondary DNA strand displacement, it ispreferred that a sufficient concentration of secondary DNA stranddisplacement primer be used to obtain sufficiently rapid priming of thegrowing TS-DNA strand to outcompete any remaining unligated OCPs and gapoligonucleotides that might be present for binding to TS-DNA. Ingeneral, this is accomplished when the secondary DNA strand displacementprimer is in very large excess compared to the concentration ofsingle-stranded sites for hybridization of the secondary DNA stranddisplacement primer on TS-DNA. Optimization of the concentration ofsecondary DNA strand displacement primer can be aided by analysis ofhybridization kinetics using methods such as those described by Youngand Anderson, "Quantitative analysis of solution hybridization" inNucleic Acid Hybridization: A Practical Approach (IRL Press, 1985) pages47-71. For example, assuming that φ29 DNA polymerase is used as therolling circle DNA polymerase, TS-DNA is generated at a rate of about 53nucleotides per second, and the rolling circle DNA polymerase generatesapproximately 10 copies of the amplification target circle in 19seconds. Analysis of the theoretical solution hybridization kinetics foran OCP driver DNA (unligated OCP) present at a concentration of 80 nM (atypical concentration used for a LM-RCA ligation operation), and thetheoretical solution hybridization kinetics for a secondary DNA stranddisplacement primer driver DNA present at a concentration of 800 nM,indicates that the secondary DNA strand displacement primer will bind tothose 10 copies within 30 seconds, while unligated OCP will hybridize toless than one site in 30 seconds (8% of sites filled). If theconcentration of DNA polymerase is relatively high (for this example, inthe range of 100 to 1000 nM), the polymerase will initiate DNA synthesisat each available 3' terminus on the hybridized secondary DNA stranddisplacement primers, and these elongating TS-DNA-2 molecules will blockany hybridization by the unligated OCP molecules. Alternatively, theefficiency of secondary DNA strand displacement can be improved by theremoval of unligated open circle probes and gap oligonucleotides priorto amplification of the TS-DNA. In secondary DNA strand displacement, itis preferred that the concentration of secondary DNA strand displacementprimer generally be from 500 nM to 5000 nM, and most preferably from 700nM to 1000 nM.

As a secondary DNA strand displacement primer is elongated, the DNApolymerase will run into the 5' end of the next hybridized secondary DNAstrand displacement molecule and will displace its 5' end. In thisfashion a tandem queue of elongating DNA polymerases is formed on theTS-DNA template. As long as the rolling circle reaction continues, newsecondary DNA strand displacement primers and new DNA polymerases areadded to TS-DNA at the growing end of the rolling circle. The generationof TS-DNA-2 and its release into solution by strand displacement isshown diagrammatically in FIG. 11.

Generally, secondary DNA strand displacement can be performed by,simultaneous with or following RCA, mixing a secondary DNA stranddisplacement primer with the polymerase-ATC mixture, and incubating thepolymerase-ATC mixture under conditions that promote both hybridizationbetween the tandem sequence DNA and the secondary DNA stranddisplacement primer, and replication of the tandem sequence DNA, wherereplication of the tandem sequence DNA results in the formation ofsecondary tandem sequence DNA.

When secondary DNA strand displacement is carried out in the presence ofa tertiary DNA strand displacement primer, an exponential amplificationof TS-DNA sequences takes place. This special and preferred mode ofsecondary DNA strand displacement is referred to as strand displacementcascade amplification (SDCA). In SDCA, illustrated in FIG. 13, asecondary DNA strand displacement primer primes replication of TS-DNA toform TS-DNA-2, as described above. The tertiary DNA strand displacementprimer strand can then hybridize to, and prime replication of, TS-DNA-2to form TS-DNA-3. Strand displacement of TS-DNA-3 by the adjacent,growing TS-DNA-3 strands makes TS-DNA-3 available for hybridization withsecondary DNA strand displacement primer. This results in another roundof replication resulting in TS-DNA-4 (which is equivalent to TS-DNA-2).TS-DNA-4, in turn, becomes a template for DNA replication primed bytertiary DNA strand displacement primer. The cascade continues thismanner until the reaction stops or reagents become limiting. Thisreaction amplifies DNA at an almost exponential rate, although kineticsare not truly exponential because there are stochastically distributedpriming failures, as well as steric hindrance events related to thelarge size of the DNA network produced during the reaction. In apreferred mode of SDCA, the rolling circle replication primer serves asthe tertiary DNA strand displacement primer, thus eliminating the needfor a separate primer. For this mode, the rolling circle replicationprimer should be used at a concentration sufficiently high to obtainrapid priming on the growing TS-DNA-2 strands. To optimize theefficiency of SDCA, it is preferred that a sufficient concentration ofsecondary DNA strand displacement primer and tertiary DNA stranddisplacement primer be used to obtain sufficiently rapid priming of thegrowing TS-DNA strand to outcompete TS-DNA for binding to itscomplementary TS-DNA, and, in the case of secondary DNA stranddisplacement primer, to outcompete any remaining unligated OCPs and gapoligonucleotides that might be present for binding to TS-DNA. Ingeneral, this is accomplished when the secondary DNA strand displacementprimer and tertiary DNA strand displacement primer are both in verylarge excess compared to the concentration of single-stranded sites forhybridization of the DNA strand displacement primers on TS-DNA. Forexample, it is preferred that the secondary DNA strand displacementprimer is in excess compared to the concentration of single-strandedsecondary DNA strand displacement primer complement sites on TS-DNA,TS-DNA-3, TS-DNA-5, and so on. In the case of tertiary DNA stranddisplacement primer, it is preferred that the tertiary DNA stranddisplacement primer is in excess compared to the concentration ofsingle-stranded tertiary DNA strand displacement primer complement siteson TS-DNA-2, TS-DNA-4, TS-DNA-6, and so on. Such an excess generallyresults in a primer hybridizing to its complement in TS-DNA beforeamplified complementary TS-DNA can hybridize. Optimization of primerconcentrations can be aided by analysis of hybridization kinetics (Youngand Anderson). In a strand displacement cascade amplification, it ispreferred that the concentration of both secondary and tertiary DNAstrand displacement primers generally be from 500 nM to 5000 nM, andmost preferably from 700 nM to 1000 nM.

As in the case of secondary DNA strand displacement primers, if theconcentration of DNA polymerase is sufficiently high, the polymerasewill initiate DNA synthesis at each available 3' terminus on thehybridized tertiary DNA strand displacement primers, and theseelongating TS-DNA-3 molecules will block any hybridization by TS-DNA-2.As a tertiary DNA strand displacement primer is elongated to formTS-DNA-3, the DNA polymerase will run into the 5' end of the nexthybridized tertiary DNA strand displacement primer molecule and willdisplace its 5' end. In this fashion a tandem queue of elongating DNApolymerases is formed on the TS-DNA-2 template. As long as the reactioncontinues, new rolling circle replication primers and new DNApolymerases are added to TS-DNA-2 at the growing ends of TS-DNA-2. Thishybridization/replication/strand displacement cycle is repeated withhybridization of secondary DNA strand displacement primers on thegrowing TS-DNA-3. The cascade of TS-DNA generation, and their releaseinto solution by strand displacement is shown diagrammatically in FIG.13.

Generally, strand displacement cascade amplification can be performedby, simultaneous with, or following, RCA, mixing a secondary DNA stranddisplacement primer and a tertiary DNA strand displacement primer withthe polymerase-ATC mixture, and incubating the polymerase-ATC mixtureunder conditions that promote hybridization between the tandem sequenceDNA and the secondary DNA strand displacement primer, replication of thetandem sequence DNA--where replication of the tandem sequence DNAresults in the formation of secondary tandem sequence DNA--hybridizationbetween the secondary tandem sequence DNA and the tertiary DNA stranddisplacement primer, and replication of secondary tandem sequenceDNA--where replication of the secondary tandem sequence DNA results information of tertiary tandem sequence DNA (TS-DNA-3).

An example of the amplification yield generated by a strand displacementcascade amplification can be roughly estimated as follows. A rollingcircle reaction that proceeds for 35 minutes at 53 nucleotides persecond will generate 1236 copies of a 90 nucleotide amplification targetcircle. Thus, TS-DNA-1 contains 1236 tandem repeats. As these 1236tandem repeats grow, priming and synthesis with secondary DNA stranddisplacement primers can generate at least 800 TS-DNA-2 molecules,taking into account delays and missed priming events. These newmolecules will have lengths linearly distributed in the range of 1 to799 repeats. Next, priming events on TS-DNA-2 by tertiary DNA stranddisplacement primers can generate at least 500 TS-DNA-3 molecules,taking into account delays and missed priming events, and these newmolecules will have lengths linearly distributed in the range of 1 to499 repeats. Then, priming events on TS-DNA-3 by secondary DNA stranddisplacement primers can generate at least 300 TS-DNA-4 molecules,taking into account delays and missed priming events, and these newmolecules will have lengths linearly distributed in the range of 1 to299 repeats. A conservative overall amplification yield, calculated asthe product of only these four amplification levels, is estimated to be1.86×10¹⁰ repeats of the original OCP or ATC. Thus, SDCA is capable ofextremely high amplification yields in an isothermal 35-minute reaction.

Secondary DNA strand displacement can also be carried out sequentially.Following a first round of secondary DNA strand displacement, a tertiaryDNA strand displacement primer can be mixed with the polymerase-ATCmixture, and the polymerase-ATC mixture can be incubated underconditions that promote hybridization between the secondary tandemsequence DNA and the tertiary DNA strand displacement primer, andreplication of secondary tandem sequence DNA, where replication of thesecondary tandem sequence DNA results in formation of tertiary tandemsequence DNA (TS-DNA-3). This round of strand displacement replicationcan be referred to as tertiary DNA strand displacement. However, allrounds of strand displacement replication following rolling circlereplication can also be referred to collectively as secondary DNA stranddisplacement.

A modified form of secondary DNA strand displacement results inamplification of TS-DNA and is referred to as opposite strandamplification (OSA). OSA is the same as secondary DNA stranddisplacement except that a special form of rolling circle replicationprimer is used that prevents it from hybridizing to TS-DNA-2. This canbe accomplished in a number of ways. For example, the rolling circlereplication primer can have an affinity tag coupled to itsnon-complementary portion allowing the rolling circle replication primerto be removed prior to secondary DNA strand displacement. Alternatively,remaining rolling circle replication primer can be crippled followinginitiation of rolling circle replication. One preferred form of rollingcircle replication primer for use in OSA is designed to form a hairpinthat contains a stem of perfectly base-paired nucleotides. The stem cancontain 5 to 12 base pairs, most preferably 6 to 9 base pairs. Such ahairpin-forming rolling circle replication primer is a poor primer atlower temperature (less than 40° C.) because the hairpin structureprevents it from hybridizing to complementary sequences. The stem shouldinvolve a sufficient number of nucleotides in the complementary portionof the rolling circle replication primer to interfere with hybridizationof the primer to the OCP or ATC. Generally, it is preferred that a steminvolve 5 to 24 nucleotides, and most preferably 6 to 18 nucleotides, ofthe complementary portion of a rolling circle replication primer. Arolling circle replication primer where half of the stem involvesnucleotides in the complementary portion of the rolling circlereplication primer and the other half of the stem involves nucleotidesin the non-complementary portion of the rolling circle replicationprimer is most preferred. Such an arrangement eliminates the need forself-complementary regions in the OCP or ATC when using ahairpin-forming rolling circle replication primer.

When starting the rolling circle replication reaction, secondary DNAstrand displacement primer and rolling circle replication primer areadded to the reaction mixture, and the solution is incubated briefly ata temperature sufficient to disrupt the hairpin structure of the rollingcircle replication primer but to still allow hybridization to the primercomplement portion of the amplification target circle (typically greaterthan 50° C.). This incubation permits the rolling circle replicationprimer to hybridize to the primer complement portion of theamplification target circle. The solution is then brought to the propertemperature for rolling circle replication, and the rolling circle DNApolymerase is added. As the rolling circle reaction proceeds, TS-DNA isgenerated, and as the TS-DNA grows in length, the secondary DNA stranddisplacement primer rapidly initiates DNA synthesis with multiple stranddisplacement reactions on TS-DNA. These reactions generate TS-DNA-2,which is complementary to the TS-DNA. While TS-DNA-2 contains sequencescomplementary to the rolling circle replication primer, the primer isnot able to hybridize nor prime efficiently at the reaction temperaturedue to its hairpin structure at this temperature. Thus, there is nofurther priming by the rolling circle replication primer and the onlyproducts generated are TS-DNA and TS-DNA-2. The reaction comes to a haltas rolling circle amplification stops and TS-DNA becomes completelydouble-stranded. In the course of the reaction, an excess ofsingle-stranded TS-DNA-2 is generated.

Another form of rolling circle replication primer useful in OSA is achimera of DNA and RNA. In this embodiment, the rolling circle primerhas deoxyribonucleotides at its 3' end and ribonucleotides in theremainder of the primer. It is preferred that the rolling circlereplication primer have five or six deoxyribonucleotides at its 3' end.By making part of the rolling circle replication primer withribonucleotide, the primer can be selectively degraded by RNAse H whenit is hybridized to DNA. Such hybrids form during OSA as TS-DNA-2 issynthesized. The deoxyribonucleotides at the 3' end allow the rollingcircle DNA polymerase to initiate rolling circle replication. RNAse Hcan then be added to the OSA reaction to prevent priming of TS-DNA-2replication.

An example of the amplification yield generated by OSA can be roughlyestimated as follows. A rolling circle reaction that proceeds for 45minutes at 53 nucleotides per second will generate tandem 1590 copies ofa 90 nucleotide amplification target circle. Thus, TS-DNA-1 contains1590 tandem repeats. As these 1590 tandem repeats grow, priming anddisplacement reactions with secondary DNA strand displacement primerswill generate and release up to 1400 TS-DNA-2 molecules, and those newmolecules will have lengths linearly distributed in the range of 1 to1399 repeats. Calculations indicate that after 45 minutes,single-stranded TS-DNA-2 exceeds the amount of TS-DNA by a factor ofabout 700. OSA is useful for generating single-stranded DNA thatcontains the reverse complement of the target sequence. Overallamplification can be of the order of one million fold.

If secondary DNA strand displacement is used with a ligated OCP,unligated OCPs and gap oligonucleotides may be removed prior to rollingcircle replication to eliminate competition between unligated OCPs andgap oligonucleotides and the secondary DNA strand displacement primerfor hybridization to TS-DNA. An exception would be when secondary DNAstrand displacement is used in conjunction with gap-filling LM-RCA, asdescribed below. Alternatively, the concentration of the secondary DNAstrand displacement primer can be made sufficiently high so that itoutcompetes unligated OCP for hybridization to TS-DNA. This allowssecondary DNA strand displacement to be performed without removal ofunligated OCPs.

The DNA generated by secondary DNA strand displacement can be labeledand/or detected using the same labels, labeling methods, and detectionmethods described for use with TS-DNA. Most of these labels and methodsare adaptable for use with nucleic acids in general. A preferred methodof labeling the DNA is by incorporation of labeled nucleotides duringsynthesis.

4. Multiple Ligation Cycles

Using a thermostable DNA ligase, such as AMPLIGASE® (EpicentreTechnologies, Inc.), the open circle probe ligation reaction may becycled a number of times between a annealing temperature (55° C.) and amelting temperature (96° C.). This cycling will produce multipleligations for every target sequence present in the sample. For example,8 cycles of ligation would provide and approximate 6-fold increase inthe number of ligated circles. A preferred cycling protocol is 96° C.for 2 seconds, 55° C. for 2 seconds, and 60° C. for 70 seconds in aPerkin Elmer 9600 thermal cycler. If the number of cycles is kept small,the linearity of the amplification response should not be compromised.

The expected net amplification yield using eight ligation cycles,secondary fluorescent tags, and array hybridization can be calculated asshown below.

    ______________________________________                                        Ligation cycling yield:                                                                              6                                                        OSA yield 1,000,000                                                           number of fluorescent tags/circle 5                                           20% array hybridization yield 0.2                                             Net yield = 6 × 1,000,000 × 5 × 0.2 = 6,000,000                                   100 target molecules × 6,000,000 = 6                                   × 10.sup.8                                         fluors bound on the surface                                                 ______________________________________                                    

5. Transcription Following RCA (RCT)

Once TS-DNA is generated using RCA, further amplification can beaccomplished by transcribing the TS-DNA from promoters embedded in theTS-DNA. This combined process, referred to as rolling circle replicationwith transcription (RCT), or ligation mediated rolling circlereplication with transcription (LM-RCT), requires that the OCP or ATCfrom which the TS-DNA is made have a promoter portion in its spacerregion. The promoter portion is then amplified along with the rest ofthe OCP or ATC resulting in a promoter embedded in each tandem repeat ofthe TS-DNA (FIG. 8). Since transcription, like rolling circleamplification, is a process that can go on continuously (withre-initiation), multiple transcripts can be produced from each of themultiple promoters present in the TS-DNA. RCT effectively adds anotherlevel of amplification of ligated OCP sequences.

Generally, RCT can be accomplished by performing RCA to produce TS-DNAin a polymerase-OCP mixture or polymerase-ATC mixture, and then mixingRNA polymerase with the polymerase-OCP mixture or polymerase-ATCmixture, resulting in a transcription mixture, and incubating thetranscription mixture under conditions promoting transcription of thetandem sequence DNA. The OCP or ATC must include the sequence of apromoter for the RNA polymerase (a promoter portion) in its spacerregion for RCT to work. The transcription step in RCT generally can beperformed using established conditions for in vitro transcription of theparticular RNA polymerase used. Preferred conditions are described inthe Examples. Alternatively, transcription can be carried outsimultaneously with rolling circle replication. This is accomplished bymixing RNA polymerase with the polymerase-OCP mixture or polymerase-ATCmixture prior to incubating the mixture for rolling circle replication.For simultaneous rolling circle replication and transcription therolling circle DNA polymerase and RNA polymerase must be active in thesame conditions. Such conditions can be optimized in order to balanceand/or maximize the activity of both polymerases. It is not necessarythat the polymerase achieve their maximum activity, a balance betweenthe activities is preferred. Transcription can follow any DNAreplication operation described herein, such as RCA, LM-RCA, nestedLM-RCA, secondary DNA strand displacement, or strand displacementcascade amplification.

The transcripts generated in RCT can be labeled and/or detected usingthe same labels, labeling methods, and detection methods described foruse with TS-DNA. Most of these labels and methods are adaptable for usewith nucleic acids in general. A preferred method of labeling RCTtranscripts is by direct labeling of the transcripts by incorporation oflabeled nucleotides, most preferably biotinylated nucleotides, duringtranscription.

6. Gap-Filling Ligation

The gap space formed by an OCP hybridized to a target sequence isnormally occupied by one or more gap oligonucleotides as describedabove. Such a gap space may also be filled in by a gap-filling DNApolymerase during the ligation operation. As an alternative, the gapspace can be partially bridged by one or more gap oligonucleotides, withthe remainder of the gap filled using DNA polymerase. This modifiedligation operation is referred to herein as gap-filling ligation and isthe preferred form of the ligation operation. The principles andprocedure for gap-filling ligation are generally analogous to thefilling and ligation performed in gap LCR (Wiedmann et al., PCR Methodsand Applications (Cold Spring Harbor Laboratory Press, Cold SpringHarbor Laboratory, NY, 1994) pages S51-S64; Abravaya et al., NucleicAcids Res., 23(4):675-682 (1995); European Patent Application EP0439182(1991)). In the case of LM-RCA, the gap-filling ligation operation issubstituted for the normal ligation operation. Gap-filling ligationprovides a means for discriminating between closely related targetsequences. An example of this is described in Example 3. Gap-fillingligation can be accomplished by using a different DNA polymerase,referred to herein as a gap-filling DNA polymerase. Suitable gap-fillingDNA polymerases are described above. Alternatively, DNA polymerases ingeneral can be used to fill the gap when a stop base is used. The use ofstop bases in the gap-filling operation of LCR is described in EuropeanPatent Application EP0439182. The principles of the design of gaps andthe ends of flanking probes to be joined, as described in EP0439182, isgenerally applicable to the design of the gap spaces and the ends oftarget probe portions described herein.

To prevent interference of the gap-filling DNA polymerase with rollingcircle replication, the gap-filling DNA polymerase can be removed byextraction or inactivated with a neutralizing antibody prior toperforming rolling circle replication. Such inactivation is analogous tothe use of antibodies for blocking Taq DNA polymerase prior to PCR(Kellogg et al., Biotechniques 16(6): 1134-1137 (1994)).

Gap-filling ligation is also preferred because it is highly compatiblewith exponential amplification of OCP sequences similar to the stranddisplacement cascade amplification (SDCA) as described above. As TS-DNAis formed during rolling circle replication, unligated OCP moleculespresent in the reaction hybridize to TS-DNA, leaving gap spaces betweenevery OCP repeat. The hybridized OCP molecules serve as primers forsecondary DNA synthesis.

Generally, gap-filling LM-RCA can be performed by, in an LM-RCAreaction, (1) using a target sequence with a central region locatedbetween a 5' region and a 3' region, and an OCP where neither the lefttarget probe portion of the open circle probe nor the right target probeportion of the open circle probe is complementary to the central regionof the target sequence, and (2) mixing gap-filling DNA polymerase withthe OCP-target sample mixture.

7. Ligation Mediated Rolling Circle Amplification with CombinatorialMulticolor Coding

A preferred form of rolling circle amplification involving multiplexdetection is Ligation Mediated Rolling Circle Amplification withCombinatorial Multicolor Coding (LM-RCA-CMC), which is a combination ofLM-RCA and CMC, both as described above. In LM-RCA-CMC, open circleprobes and corresponding gap oligonucleotides are designed for thedetection of a number of distinct target sequences. DNA samples to betested are incorporated into a solid-state sample, as described above.The solid-state substrate is preferably a glass slide and thesolid-state sample preferably incorporates up to 256 individual targetor assay samples arranged in dots. Multiple solid-state samples can beused to either test more individual samples, or to increase the numberof distinct target sequences to be detected. In the later case, eachsolid-state sample has an identical set of sample dots, and LM-RCA willbe carried out using a different set of open circle probes and gapoligonucleotides, collectively referred to as a probe set, for eachsolid-state sample. This allows a large number of individuals and targetsequences to be assayed in a single assay. By using up to six differentlabels, combinatorial multicolor coding allows up to 63 distinct targetsto be detected on a single solid-state sample. When using multiplesolid-state substrates and performing LM-RCA with a different set ofOCPs and gap oligonucleotides for each solid-state substrate, the samelabels can be used with each solid-state sample (although differencesbetween OCPs in each set may require the use of different detectionprobes), For example, 10 replica slides, each with 256 target sampledots, can be subjected to LM-RCA using 10 different sets of OCPs and gapoligonucleotides, where each set is designed for combinatorialmulticolor coding of 63 targets. This result in an assay for detectionof 630 different target sequences. Where two or more different targetsequences are closely spaced in the DNA of the target or assay sample(for example, when multiple closely spaced mutations of the same geneare targets), it is preferred that the OCPs and gap oligonucleotides foreach of the closely spaced target sequences be placed in a differentprobe set. For this purpose, it is considered that target sequenceswithin 20 nucleotides of each other on a DNA molecule in a target orassay sample are closely spaced. It is not required that multipletargets within the same gene be detected with a different probe set. Itis merely preferred that closely spaced target sequences, as definedabove, be separately probed.

After rolling circle amplification, a cocktail of detection probes isadded, where the cocktail contains color combinations that are specificfor each OCP. The design and combination of such detection probes foruse in combinatorial multicolor coding is described above. It ispreferred that the OCPs be designed with combinatorially coded detectiontags to allow use of a single set of singly labeled detection probes. Itis also preferred that collapsing detection probes be used. As describedabove, collapsing probes contain two complementary portions. This allowseach detection probe to hybridize to two detection tags in TS-DNA. Inthis way, the detection probe forms a bridge between different parts ofthe TS-DNA. The combined action of numerous collapsing detection probeshybridizing to TS-DNA will be to form a collapsed network ofcross-linked TS-DNA. Collapsed TS-DNA occupies a much smaller volumethan free, extended TS-DNA, and includes whatever detection labelpresent on the detection probe. This result is a compact and discretedetectable signal for each TS-DNA. Probe binding will, upon collapse,trap a unique combination of colors that was designed a priory on thebasis of each probe sequence.

As discussed above, rolling circle amplification can be engineered toproduce TS-DNA of different lengths for different OCPs. Such productscan be distinguish simply on the basis of the size of the detectionsignal they generate. Thus, the same set of detection probes could beused to distinguish two different sets of generated TS-DNA. In thisscheme, two different TS-DNAs, each of a different size class butassigned the same color code, would be distinguished by the size of thesignal produced by the hybridized detection probes. In this way, a totalof 126 different targets can be distinguished on a single solid-statesample using a code with 63 combinations, since the signals will come intwo flavors, low amplitude and high amplitude. Thus one could, forexample, use the low amplitude signal set of 63 probes for detection ofan oncogene mutations, and the high amplitude signal set of 63 probesfor the detection of a tumor suppressor p53 mutations.

8. Reporter Binding Agent Unimolecular Rolling Amplification

Reporter Binding Agent Unimolecular Rolling Amplification (RBAURA) is aform of RCA where a reporter binding agent provides the rolling circlereplication primer for amplification of an amplification target circle.In RBAURA, the oligonucleotide portion of the reporter binding agentserves as a rolling circle replication primer. RBAURA allows RCA toproduce an amplified signal (that is, TS-DNA) based on association ofthe reporter binding agent to a target molecule. The specific primersequence that is a part of the reporter binding agent provides the linkbetween the specific interaction of the reporter binding agent to atarget molecule (via the affinity portion of the reporter binding agent)and RCA. In RBAURA, once the reporter binding agent is associated with atarget molecule, an amplification target circle is hybridized to therolling circle replication primer sequence of the reporter bindingagent, followed by amplification of the ATC by RCA. The resulting TS-DNAincorporates the rolling circle replication primer sequence of thereporter binding agent at one end, thus anchoring the TS-DNA to the siteof the target molecule. RBAURA is a preferred RCA method for in situdetections. For this purpose, it is preferred that the TS-DNA iscollapsed using collapsing detection probes, biotin-antibody conjugates,or both, as described above. RBAURA can be performed using any targetmolecule. Preferred target molecules are nucleic acids, includingamplified nucleic acids such as TS-DNA and amplification target circles,antigens and ligands. Examples of the use of such target molecules areillustrated in FIGS. 25A to 29B. Peptide Nucleic Acid Probe UnimolecularRolling Amplification (PNAPURA) and Locked Antibody Unimolecular RollingAmplification (LAURA), described below, are preferred forms of RBAURA.

(a) Peptide Nucleic Acid Probe Unimolecular Rolling Amplification

In PNAPURA, chimeric PNA:DNA molecules are used as reporter bindingprobes, referred to as PNA reporter binding probes. The oligonucleotideportion of the PNA reporter binding agent serves as a rolling circlereplication primer. The affinity portion of the PNA reporter bindingprobe is a peptide nucleic acid, preferably 12 to 20 nucleotide bases inlength and more preferably 15 to 18 bases in length, designed tohybridize to a target nucleic acid sequence of interest. In PNAPURA, thePNA reporter binding probe is first allowed to hybridize to a targetsequence (illustrated in FIG. 25A). Once the PNA reporter binding probeis hybridized to a target sequence, an amplification target circle ishybridized to the rolling circle replication primer sequence of the PNAreporter binding probe (illustrated in FIG. 25B), followed byamplification of the ATC by RCA. The resulting TS-DNA incorporates therolling circle replication primer sequence of the PNA reporter bindingprobe at one end, thus anchoring the TS-DNA to the site of the targetmolecule. Reporter binding agents having any form of affinity portioncan be used in a similar manner.

PNAPURA is preferably performed with a solid-state substrate and incombination with CMC. For this purpose, DNA samples to be tested areincorporated into a solid-state sample, as described above. Thesolid-state substrate is preferably a glass slide and the solid-statesample preferably incorporates up to 256 individual target or assaysamples arranged in dots. Multiple solid-state samples can be used toeither test more individual samples, or to increase the number ofdistinct target sequences to be detected. In the later case, eachsolid-state sample has an identical set of samples dots, and PNAPURAwill be carried out using a different set of PNA reporter binding probesand amplification target circles, collectively referred to as a probeset, for each solid-state sample. This allows a large number ofindividuals and target sequences to be assayed in a single assay. Byusing up to six different labels, combinatorial multicolor coding allowsup to 63 distinct targets to be detected on a single solid-state sample.When using multiple solid-state substrates and performing PNAPURA with adifferent set of PNA reporter binding probes and amplification targetcircles for each solid-state substrate, the same labels can be used witheach solid-state sample (although differences between ATCs in each setmay require the use of different detection probes). For example, 10replica slides, each with 256 target sample dots, can be subjected toPNAPURA using 10 different sets of PNA reporter binding probes andamplification target circles, where each set is designed forcombinatorial multicolor coding of 63 targets. This results in an assayfor detection of 630 different target sequences. Where two or moredifferent target sequences are closely spaced in the DNA of the targetor assay sample (for example, when multiple closely spaced mutations ofthe same gene are targets), it is preferred that the PNA reporterbinding probe for each of the closely spaced target sequences be placedin a different probe set. For this purpose, it is considered that targetsequences within 20 nucleotides of each other on a DNA molecule in atarget or assay sample are closely spaced. It is not required thatmultiple targets within the same gene be detected with a different probeset. It is merely preferred that closely spaced target sequences, asdefined above, be separately probed.

After rolling circle amplification, a cocktail of detection probes isadded, where the cocktail contains color combinations that are specificfor each ATC. The design and combination of such detection probes foruse in combinatorial multicolor coding is described above. It ispreferred that the ATCs be designed with combinatorially coded detectiontags to allow use of a single set of singly labeled detection probes. Itis also preferred that collapsing detection probes be used. As describedabove, collapsing probes contain two complementary portions. This allowseach detection probe to hybridize to two detection tags in TS-DNA. Inthis way, the detection probe forms a bridge between different parts ofthe TS-DNA. The combined action of numerous collapsing detection probeshybridizing to TS-DNA will be to form a collapsed network ofcross-linked TS-DNA. Collapsed TS-DNA occupies a much smaller volumethan free, extended TS-DNA, and includes whatever detection labelpresent on the detection probe. This result is a compact and discretedetectable signal for each TS-DNA. Probe binding will, upon collapse,trap a unique combination of colors that was designed a priory on thebasis of each probe sequence.

(b) Locked Antibody Unimolecular Rolling Amplification

In LAURA, a covalently coupled antibody and oligonucleotide is used as areporter binding agent. The oligonucleotide portion of the reporterbinding agent serves as a rolling circle replication primer. Theaffinity portion of the reporter binding agent is an antibody specificfor a target molecule of interest. The reporter binding agent isconjugated to the target molecule as in a conventional immunoassay(illustrated in FIG. 29A). Unlike conventional immunoassays, detectionof this interaction is mediated by rolling circle amplification. Afterconjugation and washing, the immune complexes are fixed in place with asuitable fixation reaction (for example, methanol-acetic acid) toimmobilize the antibody. As in conventional immunoassays, unconjugatedantibodies (in this case, in the form of reporter binding agents) areremoved by washing. Once the reporter binding agent is conjugated to atarget molecule, an amplification target circle is hybridized to therolling circle replication primer sequence of the reporter binding agent(illustrated in FIG. 29B), followed by amplification of the ATC by RCA.The resulting TS-DNA incorporates the rolling circle replication primersequence of the reporter binding agent at one end, thus anchoring theTS-DNA to the site of the target molecule.

In a variant of this method, the oligonucleotide portion of the reporterbinding agent can be a peptide nucleic acid, instead of DNA. Afterfixation of the reporter binding agent to the target molecule, the PNAcan be hybridized an oligonucleotide that contains a portioncomplementary to the PNA, referred to as the complementary portion, anda portion that remains single stranded, referred to as the primerportion. The primer portion can then be used as a rolling circle primerin LAURA as described above.

LAURA is preferably performed with a solid-state substrate and incombination with CMC. For this purpose, DNA samples to be tested areincorporated into a solid-state sample, as described above. Thesolid-state substrate is preferably a glass slide and the solid-statesample preferably incorporates up to 256 individual target or assaysamples arranged in dots. Multiple solid-state samples can be used toeither test more individual samples, or to increase the number ofdistinct target sequences to be detected. In the later case, eachsolid-state sample has an identical set of samples dots, and LAURA willbe carried out using a different set of reporter binding agents andamplification target circles, collectively referred to as a probe set,for each solid-state sample. This allows a large number of individualsand target sequences to be assayed in a single assay. By using up to sixdifferent labels, combinatorial multicolor coding allows up to 63distinct targets to be detected on a single solid-state sample. Whenusing multiple solid-state substrates and performing LAURA with adifferent set of reporter binding agents and amplification targetcircles for each solid-state substrate, the same labels can be used witheach solid-state sample (although differences between ATCs in each setmay require the use of different detection probes). For example, 10replica slides, each with 256 target sample dots, can be subjected toLAURA using 10 different sets of reporter binding agents andamplification target circles, where each set is designed forcombinatorial multicolor coding of 63 targets. This results in an assayfor detection of 630 different target sequences. Where two or moredifferent target sequences are closely spaced in the DNA of the targetor assay sample, it is preferred that the PNA reporter binding probe foreach of the closely spaced target sequences be placed in a differentprobe set, as discussed above.

After rolling circle amplification, a cocktail of detection probes isadded, where the cocktail contains color combinations that are specificfor each ATC. The design and combination of such detection probes foruse in combinatorial multicolor coding is described above. It ispreferred that the ATCs be designed with combinatorially coded detectiontags to allow use of a single set of singly labeled detection probes. Itis also preferred that collapsing detection probes be used. As describedabove, collapsing probes contain two complementary portions. This allowseach detection probe to hybridize to two detection tags in TS-DNA. Inthis way, the detection probe forms a bridge between different parts ofthe TS-DNA. The combined action of numerous collapsing detection probeshybridizing to TS-DNA will be to form a collapsed network ofcross-linked TS-DNA. Collapsed TS-DNA occupies a much smaller volumethan free, extended TS-DNA, and includes whatever detection labelpresent on the detection probe. This result is a compact and discretedetectable signal for each TS-DNA. Probe binding will, upon collapse,trap a unique combination of colors that was designed a priory on thebasis of each probe sequence.

9. Primer Extension Sequencing

Following amplification, the nucleotide sequence of the amplifiedsequences can be determined either by conventional means or by primerextension sequencing of amplified target sequence. Primer extensionsequencing is also referred herein as chain terminating primer extensionsequencing. A preferred form of chain terminating primer extensionsequencing, referred to herein as single nucleotide primer extensionsequencing, involves the addition of a single chain-terminatingnucleotide to a primer (no other nucleotides are added). This form ofprimer extension sequencing allows interrogation (and identification) ofthe nucleotide immediately adjacent to the region to which the primer ishybridized. Two preferred modes of single nucleotide primer extensionsequencing are disclosed.

(a) Unimolecular Segment Amplification and Sequencing

Unimolecular Segment Amplification and Sequencing (USA-SEQ) involvesinterrogation of a single nucleotide in an amplified target sequence byincorporation of a specific and identifiable nucleotide based on theidentity of the interrogated nucleotide. In Unimolecular SegmentAmplification and Sequencing (USA-SEQ) individual target molecules areamplified by rolling circle amplification. Following amplification, aninterrogation primer is hybridized immediately 5' of the base in thetarget sequence to be interrogated, and a single chain-terminatingnucleotide is added to the end of the primer. The identity of theinterrogated base determines which nucleotide is added. By usingnucleotides with unique detection signatures (e.g. different fluorescentlabels), the identity of the interrogated base can be determined. Theinterrogation primer can be a pre-formed single molecule or it can beformed by hybridizing one or more interrogation probes to the amplifiedtarget sequences and ligating them together to form an interrogationprimer.

USA-SEQ is useful for identifying the presence of multiple distinctsequences in a mixture of target sequences. In particular, if the samplefrom which the target sequences are amplified contains different formsof the target sequence (that is, different alleles of the targetsequence), then USA-SEQ can identify not only their presence but alsoprovide information on the relative abundance of the different forms.This is possible because each TS-DNA molecule is amplified from a singletarget sequence molecule and each TS-DNA molecule can be individuallydetected.

Primer extension sequencing can be performed generally as follows. Afteramplification of a target nucleic acid sequence using any of the rollingcircle amplification techniques disclosed herein, an interrogationprimer is hybridized to the amplified nucleic acid (for example, toTS-DNA). The mixture of amplified nucleic acid and interrogation primeris referred to as an interrogation mixture. The interrogation primer isdesigned to hybridize adjacent to (that is 3' of) the nucleotide in theTS-DNA that is to be interrogated (that is, sequenced). Of course, sincethe target sequence is repeated numerous times in a TS-DNA molecule,numerous interrogation probes will hybridize to a single TS-DNAmolecule. Next, at least two differently labeled chain terminatingnucleotides and DNA polymerase are added to the interrogation mixture.This results in addition of a single nucleotide to the end of theinterrogation primer, the identity of which is based on the identity ofthe interrogated nucleotide (that is, the first template nucleotideafter the end of the interrogation primer). Finally, the identity of thenucleotide incorporated for each TS-DNA molecule is determined byfluorescence microscopy. For this purpose, it is preferred that theTS-DNA be collapsed prior to detection of the incorporated nucleotide.Example 9 describes an example of the use of USA-SEQ to detect of homo-or heterozygosity at a particular nucleotide in a genetic sample. It isspecifically contemplated that primer extension sequencing can be usedto determine the identity of one or more specific nucleotides in anyamplified nucleic acid, including nucleotides derived from a targetnucleic acid, and nucleotides present as arbitrarily chosen sequences inthe spacer region of an OCP or ATC. In the later case, primer extensionsequencing can be used to distinguish or identify a specific OCP or ATCwhich has been amplified. As described elsewhere, the detection ofspecific OCPs and ATCs, from among an original pool of OCPs or ATCs,amplified based on the presence of a specific target molecule or nucleicacid is a preferred use for the disclosed amplification and detectionmethods.

Preferred chain terminating nucleotides are dideoxynucleotides. Otherknown chain terminating nucleotides (for example, nucleotides havingsubstituents at the 3' position) can also be used. Fluorescent forms ofdideoxynucleotides are known for use in conventional chain terminatingsequencing, any of which are suitable for the disclosed primer extensionsequencing. Preferred forms of fluorescent or haptenatedchain-terminating nucleotides include fluorescein-N6-ddATP,biotin-N6-ddATP, fluorescein-12-ddATP, fluorescein-12-ddCTP,fluorescein-12-ddGTP, fluorescein-12-ddUTP, lissamine-5-ddGTP,eosin-6-ddCTP, coumarin-ddUTP, tetramethylmodamine-6-ddUTP, TexasRed-5-ddATP (all available from NEN Life Sciences).

(b) Degenerate Probe Primer Extension Sequencing

Degenerate probe primer extension sequencing involves sequentialaddition of degenerate probes to an interrogation primer hybridized toamplified target sequences. Addition of multiple probes is prevented bythe presence of a removable blocking group at the 3' end. After additionof the degenerate probes, the blocking group is removed and furtherdegenerate probes can be added or, as the final operation, thenucleotide next to the end of the interrogation probe, or the last addeddegenerate probe, is interrogated as described for single nucleotideprimer extension sequencing to determine its identity. It iscontemplated that degenerate probes having any form of removable 3' endblock can be used in a primer extension sequencing procedure. Apreferred form of removable blocking group is the cage structure, asdescribed herein. In each case, conditions specific for removal of theparticular blocking structure are used as appropriate. A preferred formof amplification and degenerate probe primer extension sequencing isUnimolecular Segment Amplification and CAGE Sequencing (USA-CAGESEQ).

Primer extension sequencing using blocked degenerate probes (that is,degenerate probe primer extension sequencing, of which CAGESEQ is apreferred form) can be performed generally as follows. One or moreinterrogation probes and a plurality of degenerate probes are mixed withan DNA sample to be sequenced to form an interrogation mixture. It ispreferred that the nucleic acid to be sequenced is a nucleic acidamplified using any of the rolling circle amplification techniquesdisclosed herein. In this case it is further preferred that the nucleicacid to be sequenced is amplified form an amplification target circleformed by gap-filling ligation of an open circle probe. For degenerateprobe primer extension sequencing it is also preferred that a full setof degenerate probes, as described above, be used. The interrogationprobes are designed to hybridize to the target nucleic acid such thatthe region of the target nucleic acid to be sequenced lies past the 3'end of the interrogation probe. The interrogation mixture is incubatedunder conditions that promotes hybridization of the interrogation probeand the degenerate primers to the nucleic acid to be sequenced. Only oneof the degenerate probes will form a perfect hybrid with the nucleicacid sequence adjacent to the interrogation probe. It is preferred thatincubation conditions be chosen which will favor the formation ofperfect hybrids. Once the interrogation and degenerate probes arehybridized, the interrogation mixture is subjected to ligation. Thisjoins the interrogation probe and the degenerate primer. Finally, theblocking group present at the 3' end of the ligated degenerate probe isremoved. When using photolabile caged oligonucleotides, the cagestructure is removed by exposure to appropriate light. This makes theend of the ligated degenerate probe available for either ligation ofanother degenerate probe or primer extension. These hybridization,ligation, and block removal steps are referred to herein as a round ofdegenerate probe ligation. Additional rounds of degenerate probeligation can be performed following removal of the blocking structure.It is preferred that a set of primer extension sequencing assays beperformed, using identical samples, in which a different number ofrounds of degenerate probe ligation are performed prior to primerextension. It is also preferred that a nested set of interrogationprobes be used in a set of such a set of primer extension sequencingassays. The use of such a set of assays is illustrated in Example 10.Once all the rounds of degenerate probe ligation are performed (thusforming an interrogation primer), the interrogation mixture is subjectedto primer extension. For this, at least two differently labeled chainterminating nucleotides and DNA polymerase are added to theinterrogation mixture. This results in addition of a single nucleotideto the end of the interrogation primers, the identity of which is basedon the identity of the interrogated nucleotide (that is, the firsttemplate nucleotide after the end of the interrogation primer). Finally,the identity of the nucleotide incorporated for each interrogationprimer for each target nucleic acid is determined by fluorescencemicroscopy. For this purpose, it is preferred that the nucleic acid becollapsed prior to detection of the incorporated nucleotide.

Example 10 describes an example of USA-CAGESEQ where a nested set ofinterrogation primers are extended by sequential addition of degenerateprimers in an array of amplified nucleic acids. The principles of theprimer extension sequencing operation illustrated in this example can beanalogously applied to the use of different numbers of sample andinterrogation probes, different arrangements of samples and differentforms of blocking structures. It is contemplated that sets of assays canbe performed on arrays of sample dots (as shown in Example 10), inarrays of samples (such as in microtiter dishes), or in individualreaction vessels. In particular, the use of a multiwell dish, such as amicrotiter dish, allows multiple separate reactions on the same dish tobe easily automated. The use of multiple wells also allows completefreedom in the selection of the sample and interrogation probe in eachwell. For example, rather than performing primer extension sequencingusing five separately treated slides (as in example 10), primerextension sequencing samples and components could be arranged in anyconvenient order in the wells. Using the components of Example 10, forexample, a five well by five well array of identical nucleic acidsamples could be used where each of the wells in a given column has thesame interrogation probe. The first column of wells would have the firstinterrogation probe, the second column of wells would have the secondinterrogation probe, and so on. As in example 10, the mask would bemoved down to cover one additional row prior to each cage removal step.The resulting sequence obtained using this arrangement would be readacross and then down.

As described above, specific portions of TS-DNA or TS-RNA can besequenced using a primer extension sequencing operation. It should alsobe understood that the same primer extension sequencing procedure can beperformed on any nucleic acid molecule. For example, genomic DNA, PCRproducts, viral RNA or DNA, and cDNA samples can all be sequenced usingthe disclosed primer extension sequencing procedure. A preferred primerextension sequencing procedure for this purpose is CAGE sequencing. Forthis purpose, interrogation probes and degenerate probes are hybridizedto a nucleic acid sample of interest (rather than TS-DNA or TS-RNA),ligated, and subjected to chain-terminating primer extension, all asdescribed above in connection with USA-CAGESEQ.

D. Discrimination Between Closely Related Target Sequences

Open circle probes, gap oligonucleotides, and gap spaces can be designedto discriminate closely related target sequences, such as geneticalleles. Where closely related target sequences differ at a singlenucleotide, it is preferred that open circle probes be designed with thecomplement of this nucleotide occurring at one end of the open circleprobe, or at one of the ends of the gap oligonucleotide(s). Wheregap-filling ligation is used, it is preferred that the distinguishingnucleotide appear opposite the gap space. This allows incorporation ofalternative (that is, allelic) sequence into the ligated OCP without theneed for alternative gap oligonucleotides. Where gap-filling ligation isused with a gap oligonucleotide(s) that partially fills the gap, it ispreferred that the distinguishing nucleotide appear opposite the portionof gap space not filled by a gap oligonucleotide. Ligation of gapoligonucleotides with a mismatch at either terminus is extremelyunlikely because of the combined effects of hybrid instability andenzyme discrimination. When the TS-DNA is generated, it will carry acopy of the gap oligonucleotide sequence that led to a correct ligation.Gap oligonucleotides may give even greater discrimination betweenrelated target sequences in certain circumstances, such as thoseinvolving wobble base pairing of alleles. Features of open circle probesand gap oligonucleotides that increase the target-dependency of theligation operation are generally analogous to such features developedfor use with the ligation chain reaction. These features can beincorporated into open circle probes and gap oligonucleotides for use inLM-RCA. In particular, European Patent Application EP0439182 describesseveral features for enhancing target-dependency in LCR that can beadapted for use in LM-RCA. The use of stop bases in the gap space, asdescribed in European Patent Application EP0439182, is a preferred modeof enhancing the target discrimination of a gap-filling ligationoperation.

A preferred form of target sequence discrimination can be accomplishedby employing two types of open circle probes. These two OCPs would bedesigned essentially as shown in FIG. 2, with small modifications. Inone embodiment, a single gap oligonucleotide is used which is the samefor both target sequences, that is, the gap oligonucleotide iscomplementary to both target sequences. In a preferred embodiment, agap-filling ligation operation can be used (Example 3). Target sequencediscrimination would occur by virtue of mutually exclusive ligationevents, or extension-ligation events, for which only one of the twoopen-circle probes is competent. Preferably, the discriminatornucleotide would be located at the penultimate nucleotide from the 3'end of each of the open circle probes. The two open circle probes wouldalso contain two different detection tags designed to bind alternativedetection probes and/or address probes. Each of the two detection probeswould have a different detection label. Both open circle probes wouldhave the same primer complement portion. Thus, both ligated open circleprobes can be amplified using a single primer. Upon array hybridization,each detection probe would produce a unique signal, for example, twoalternative fluorescence colors, corresponding to the alternative targetsequences.

E. Optimization of RCA

1. Assay Background

A potential source of background signals is the formation of circularmolecules by non-target-directed ligation events. The contribution ofsuch events to background signals can be minimized using fivestrategies, alone or in combination, as follows:

(a) The use of a thermostable DNA ligase such as AMPLIGASE® (Kalin etal. (1992)) or the T. thermophilus DNA ligase (Barany (1991)) willminimize the frequency of non-target-directed ligation events becauseligation takes place at high temperature (50 to 75° C.).

(b) In the case of in situ hybridization, ligation of the open circleprobe to the target sequence permits extensive washing. This washingwill remove any circles that may have been formed by non-target-directedligation, while circles ligated on-target are impossible to removebecause they are topologically trapped (Nilsson et al. (1994)).

(c) The use of one or more gap oligonucleotides, or a combination of gapoligonucleotides and gap-filling DNA synthesis, provides additionalspecificity in the ligation event. Using a gap oligonucleotide greatlyreduces the probability of non-target-directed ligation. Particularlyfavored is the use of a gap oligonucleotide, or a gap-filling ligationoperation, coupled to a capture hybridization step where thecomplementary portion of an address probe spans the ligation junction ina highly discriminatory fashion, as shown below and in FIG. 6.complement of gap oligonucleotide(11 nucleotides) - \ /...NNNTA{GTCAGATCAGA}TANNNNN... TS-DNA || ||||.vert line.||.vertline.||| || AT CAGTCTAGTCT ATNNNNN... address probe / \ complementaryportion of address probe(15 nucleotides hybridized) }

Brackets ({}) mark sequence complementary to the gap oligonucleotide (orthe gap space when filled in). The TS-DNA shown is SEQ ID NO:10 and theaddress probe sequence shown is SEQ ID NO:4. This system can be usedwith gap oligonucleotides of any length. Where the gap between the endsof an open circle probe hybridized to a target sequence is larger thanthe desired address probe length, an address probe can be designed tooverlap just one of the junctions between the gap sequence and the opencircle probe sequence. By designing open circle probes to placediscriminating nucleotides opposite the gap space, a single OCP can beused in gap-filling LM-RCA to generate ligated open circle probes havingdifferent sequences, which depend on the target sequence.

The capture step involves hybridization of the amplified DNA to anaddress probe via a specific sequence interaction at the ligationjunction, involving the complement of the gap oligonucleotide, as shownabove. Guo et al. (1994), have shown that 15-mer oligonucleotides boundcovalently on glass slides using suitable spacers, can be used tocapture amplified DNA with reasonably high efficiency. This system canbe adapted to detection of amplified nucleic acid (TS-DNA or TS-RNA) byusing address probes to capture the amplified nucleic acid. In theexample shown above, only LM-RCA amplified DNA generated from correctligation events will be captured on the solid-state detector.

Optionally one may use additional immobilizing reagents, known in theart as capture probes (Syvanen et al., Nucleic Acids Res., 14:5037(1986)) in order to bind nucleic acids containing the target sequence toa solid surface. Suitable capture probes contain biotinylatedoligonucleotides (Langer et al. (1981)) or terminal biotin groups.Immobilization may take place before or after the ligation reaction.Immobilization serves to allow removal of unligated open circle probesas well as non-specifically ligated circles.

(d) Using ligation conditions that favor intramolecular ligation.Conditions are easily found where circular ligation of OCPs occurs muchmore frequently than tandem linear ligation of two OCPs. For example,circular ligation is favored when the temperature at which the ligationoperation is performed is near the melting temperature (T_(m)) of theleast stable of the left target probe portion and the right target probeportion when hybridized to the target sequence. When ligation is carriedout near the T_(m) of the target probe portion with the lowest T_(m),the target probe portion is at association/dissociation equilibrium. Atequilibrium, the probability of association in cis (that is, with theother target probe portion of the same OCP) is much higher than theprobability of association in trans (that is, with a different OCP).When possible, it is preferred that the target probe portions bedesigned with melting temperatures near suitable temperatures for theligation operation. The use of a thermostable ligase, however, allows awide range of ligation temperatures to be used, allowing greater freedomin the selection of target sequences.

(e) Peptide nucleic acids form extremely stable hybrids with DNA, andhave been used as specific blockers of PCR reactions (Orum et al.,Nucleic Acids Res., 21:5332-5336 (1993)). A special PNA probe, referredto herein as a PNA clamp, can be used to block rolling circleamplification of OCPs that have been ligated illegitimately (that is,ligated in the absence of target). By using one or more gapoligonucleotides during ligation, by using gap-filling ligation, or byusing a combination of gap oligonucleotides and gap-filling ligation,illegitimately ligated circles will lack the gap sequence and they canbe blocked with a PNA clamp that is complementary to the sequenceresulting from the illegitimate ligation of the 3' end and the 5' end ofthe OCP. This is illustrated in the diagram below, where the PNA clampllllrrrr is positioned exactly over the junction: llllrrrr PNA clamp||||||.vertline.| ...LLLLLLLLLLRRRRRRRRRR... ligated OCP /\ ligationsite

In this diagram, "L" and "l" represent a nucleotide in the left targetprobe portion of the OCP and its complement in the PNA clamp, and "R"and "r" represent a nucleotide in the right target probe portion of theOCP and its complement in the PNA clamp. The most preferred length for aPNA clamp is 8 to 10 nucleotides. The PNA clamp is incapable ofhybridizing to unligated OCP because it can only form four to five basepairs with either target probe portion, and it is also incapable ofhybridizing with correctly ligated OCP because a gap sequence ispresent. However, the PNA clamp will hybridize strongly withillegitimately ligated OCP, and it will block the progress of therolling circle reaction because the DNA polymerase is incapable ofdisplacing a hybridized PNA molecule. This prevents amplification ofillegitimately ligated OCPs.

2. Removing Excess Unligated OCPs

The gene 6 exonuclease of phage T7 provides a useful tool for theelimination of excess open circle probes and excess gap oligonucleotidesthat will bind to the TS-DNA or LM-RCT transcripts and interfere withits hybridization to detection probes. This exonuclease digests DNAstarting from the 5'-end of a double-stranded structure. It has beenused successfully for the generation of single-stranded DNA after PCRamplification (Holloway et al., Nucleic Acids Res. 21:3905-3906 (1993);Nikiforov et al., PCR Methods and Applications 3:285-291(1994)). In anLM-RCA assay this enzyme can be added after ligation, together with therolling circle DNA polymerase. To protect TS-DNA from degradation, therolling circle replication primer can contain 3 or 4 phosphorothioatelinkages at the 5' end, to make this molecule resistant to theexonuclease (Nikiforov et al. (1994)). The exonuclease will degradeexcess open circle probe molecules as they become associated with therolling circle DNA product. The use of this nuclease eliminates the needfor capture probes as well as the need for washing to remove excessprobes. In general, such a nuclease digestion should not be used whenperforming LM-RCT, since unligated OCPs and gap oligonucleotides areneeded to form a double-stranded transcription template with the TS-DNA.This nuclease digestion is a preferred method of eliminating unligatedOCPs and gap oligonucleotides when nested LM-RCA is to be performed.

EXAMPLES Example 1 Target-mediated Ligation of Open Circle Probes andRolling Circle Replication of Ligated Open Circle Probes

1. Ligation of Open Circle Probes

Linear oligonucleotides with 5'-phosphates are efficiently ligated byligase in the presence of a complementary target sequence. Inparticular, open circle probes hybridized to a target sequence as shownin FIG. 1, and open circle probes with gap oligonucleotides hybridizedto a target sequence as shown in as shown in FIG. 2, are readilyligated. The efficiency of such ligation can be measured by LM-RCA.

The following is an example of target-dependent ligation of an opencircle probe:

A DNA sample (target sample) is heat-denatured for 3 minutes at 95° C.,and incubated under ligation conditions (45 minutes at 60° C.) in abuffer consisting of 20 mM Tris-HCl (pH 8.2), 25 mM KCl, 10 mM MgCl₂,0.5 mM NAD, 0.05% Triton X-100, in the presence of (a) DNA ligase(AMPLIGASE®, Epicentre Technologies) at a concentration of 1 unit per 50μl, and (b) the following 5'-phosphorylated oligonucleotides:

Open circle probe (111 nucleotides):

5'-GCCTGTCCAGGGATCTGCTCAAGACTCGTCATGTCTCAGTAGCTTCTAACGGTCACAAGCTTCTAACGGTCACAAGCTTCTAACGGTCACAT GTCTGCTGCCCTCTGTATT-3'(SEQ ID NO:1)

Gap oligonucleotide:

5'-CCTT-3'

This results in hybridization of the open circle probe and gapoligonucleotide to the target sequence, if present in the target sample,and ligation of the hybridized open circle probe and gapoligonucleotide.

2. Measuring the Rate of Rolling Circle Replication

(a) On large template: 7 kb single-stranded phage M13 circle

The rate of oligonucleotide-primed rolling circle replication onsingle-stranded M13 circles mediated by any DNA polymerase can bemeasured by using the assay described by Blanco et al., J. Biol. Chem.264:8935-8940 (1989). The efficiency of primed synthesis by the φ29 DNApolymerase is stimulated about 3-fold in the presence of Gene-32protein, a single-stranded DNA binding protein.

(b) On small templates: 110-nucleotide ligated open circle probes

The rate of oligonucleotide-primed rolling circle replication onsingle-stranded small circles of 110 bases was measured using the φ29DNA polymerase generally as described in Example 2. After five minutesof incubation, the size of the DNA product is approximately 16kilobases. This size corresponds to a polymerization rate of 53nucleotides per second. The rate of synthesis with other DNA polymerasescan be measured and optimized using a similar assay, as described byFire and Xu, Proc. Natl. Acad. Sci. USA 92:4641-4645 (1995). It ispreferred that single-stranded circles of 110 nucleotides be substitutedfor the 34 nucleotide circles of Fire and Xu.

The φ29 DNA polymerase provides a rapid rate of polymerization of theφ29 rolling circle reaction on 110 nucleotide circular templates. At theobserved rate of 50 nucleotides per second, a 35 minute polymerizationreaction will produce a DNA product of approximately 105,000 bases. Thiswould yield an amplification of 954-fold over the original 110-basetemplate. Fire and Xu (1995) shows that rolling circle reactionscatalyzed by bacterial DNA polymerases may take place on very smallcircular templates of only 34 nucleotides. On the basis of the resultsof Fire and Yu, rolling circle replication can be carried out usingcircles of less than 90 nucleotides.

Example 2 Detection of a Mutant Ornithine Transcarbamylase (OTC) GeneUsing LM-RCA Followed by Transcription (LM-RCT)

This example describes detection of human DNA containing a mutant form(G to C) at position 114 of exon 9 of the ornithine transcarbamylasegene (Hata et al., J. Biochem. 103:302-308 (1988)). Human DNA for theassay is prepared by extraction from buffy coat using a standard phenolprocedure.

1. Two DNA samples (400 ng each) are heat-denatured for 4 minutes at 97°C., and incubated under ligation conditions in the presence of two5'-phosphorylated oligonucleotides, an open circle probe and one gapoligonucleotide:

Open circle probe (95 nucleotides):

5'-GAGGAGAATAAAAGTTTCTCATAAGACTCGTCATGTCTCAGCAGCTTCTAACGGTCACTAATACGACTCACTATAGGTTCTGCCTCTGGGAA CAC-3' (SEQ ID NO:5)

Gap oligonucleotide for mutant gene (8 nucleotides)

5'-TAGTGATG-3'

Gap oligonucleotide for wild type gene (8 nucleotides)

5'-TAGTGATC-3'

T4 DNA ligase (New England Biolabs) is present at a concentration of 5units per μl, in a buffer consisting of 10 mM Tris-HCl (pH 7.5), 0.20 MNaCl, 10 mM MgCl₂, 2 mM ATP. The concentration of open circle probe is80 nM, and the concentration of gap oligonucleotide is 100 nM. The totalvolume is 40 μl. Ligation is carried out for 25 minutes at 37° C.

2. 25 μl are taken from each of the above reactions and mixed with anequal volume of a buffer consisting of 50 mM Tris-HCl (pH 7.5), 10 mMMgCl₂, 1 mM DTT, 400 μM each of dTTP, dATP, dGTP, dCTP, which containsan 18-base rolling circle replication primer 5'-GCTGAGACATGACGAGTC-3'(SEQ ID NO:6), at a concentration of 0.2 μM. The φ29 DNA polymerase (160ng per 50 μl) is added and the reaction mixture is incubated for 30minutes at 30° C.

3. To the above solutions a compensating buffer is added to achieve thefollowing concentrations of reagents: 35 mM Tris-HCl (pH 8.2), 2 mMspermidine, 18 mM MgCl₂, 5 mM GMP, 1 mM of ATP, CTP, GTP, 333 μM UTP,667 μM Biotin-16-UTP (Boehringher-Mannheim), 0.03% Tween-20, 2 Units perμl of T7 RNA polymerase. The reaction is incubated for 90 minutes at 37°C.

4. One-tenth volume of 5 M NaCl is added to the above reactions, and theresulting solution is mixed with an equal volume of ExpressHyb reagent(Clontech Laboratories, Palo Alto, Calif.). Hybridization is performedby contacting the amplified RNA solution, under a cover slip, with thesurface of a glass slide (Guo et al. (1994)) containing a 2.5 mm dotwith 2×10¹¹ molecules of a covalently bound 29-mer oligonucleotide withthe sequence 5'-TTTTTTTTTTTCCAACCTCCATCACTAGT-3' (SEQ ID NO:7). The last14 nucleotides of this sequence are complementary to the amplifiedmutant gene RNA, and hence the mutant RNA binds specifically. Another2.5 mm dot on the slide surface contains 2×10¹¹ molecules of acovalently bound 29-mer oligonucleotide with the sequence5'-TTTTTTTTTTTCCAACCTCGATCACTAGT-3' (SEQ ID NO:8). The last 14nucleotides of this sequence are complementary to the amplified wildtype gene RNA, and hence the wild type RNA binds specifically. The glassslide is washed once with 2×SSPE as described (Guo et al. (1994)), thenwashed twice with 2×SSC (0.36 M sodium saline citrate), and thenincubated with fluoresceinated avidin (5 μg/ml) in 2×SSC for 20 minutesat 30° C. The slide is washed 3 times with 2×SSC and the slide-boundfluorescence is imaged at 530 nm using a Molecular Dynamics Fluorimager.

Example 3 Detection of a Mutant Ornithine Transcarbamylase (OTC) GeneUsing Gap-Filling LM-RCT

This example describes detection of human DNA containing a mutant form(G to C) at position 114 of exon 9 of the ornithine transcarbamylasegene (Hata et al. (1988)) using gap-filling LM-RCT. Human DNA for theassay is prepared by extraction from buffy coat using a standard phenolprocedure. In this example, two different open circle probes are used todetect the mutant and wild type forms of the gene. No gapoligonucleotide is used.

1. Two DNA samples (400 ng each) are heat-denatured for 4 minutes at 97°C., and incubated in the presence of one of the following5'-phosphorylated open circle probes.

Open circle probe for mutant gene (96 nucleotides):

5'-TAAAAGACTTCATCATCCATCTCATAAGACTCGTCATGTCTCAGCAGCTTCTAACGGTCACTAATACGACTCACTATAGGGGAACACTAGT GATGG-3' (SEQ ID NO:11).When this probe hybridizes to the target sequence, there is a gap spaceof seven nucleotides between the ends of the open circle probe.

Open circle probe for wild type gene (96 nucleotides):

5'-TAAAAGACTTCATCATCCATCTCATAAGACTCGTCATGTCTCAGCAGCTTCTAACGGTCACTAATACGACTCACTATAGGGGAACACTAGT GATCG-3' (SEQ ID NO:12).When this probe hybridizes to the target sequence, there is a gap spaceof seven nucleotides between the ends of the open circle probe.

Each of the OCP-target sample mixtures are incubated in anextension-ligation mixture as described by Abravaya et al. (1995). Thereaction, in a volume of 40 μl, contains 50 mM Tris-HCl (pH 7.8), 25 mMMgCl₂, 20 mM potassium acetate, 10 μM NAD, 80 nM open circle probe, 40μM dATP, 40 μM dGTP, 1 Unit Thermus flavus DNA polymerase (lacking 3'-5'exonuclease activity; MBR, Milwaukee, Wis.), and 4000 Units Thermusthermophilus DNA ligase (Abbott laboratories). The reaction is incubatedfor 60 seconds at 85° C., and 50 seconds at 60° C. in a thermal cycler.No thermal cycling is performed. This results in hybridization of theopen circle probe to the target sequence, if present, filling in of thegap space by the T. flavus DNA polymerase, and ligation by the T.thermophilus ligase. The discriminating nucleotide in the open circleprobes above is the penultimate nucleotide. T. flavus DNA polymerase isused in the reaction to match the thermal stability of the T.thermophilus ligase.

2. 25 μl are taken from each of the above reactions and mixed with anequal volume of a buffer consisting of 50 mM Tris-HCl (pH 7.5), 10 mMMgCl₂, 1 mM DTT, 400 μM each of dTTP, dATP, dGTP, dCTP; and containingthe 18-base oligonucleotide primer 5'-GCTGAGACATGACGAGTC-3' (SEQ IDNO:6), at a concentration of 0.2 μM. The φ29 DNA polymerase (160 ng per50 μl) is added and the reaction mixture is incubated for 30 minutes at30° C. to perform rolling circle amplification catalyzed by φ29 DNApolymerase. The Thermus flavus DNA polymerase does not significantlyinterfere with rolling circle replication because it has little activityat 30° C. If desired, the Thermus flavus DNA polymerase can beinactivated, prior to rolling circle replication, by adding aneutralizing antibody analogous to antibodies for blocking Taq DNApolymerase prior to PCR (Kellogg et al., Biotechniques 16(6):1134-1137(1994)).

3. To each of the above solutions are added compensating buffer toachieve the following concentrations of reagents: 35 mM Tris-HCl (pH8.2), 2 mM spermidine, 18 mM MgCl₂, 5 mM GMP, 1 mM of ATP, CTP, GTP, 333μM UTP, 667 μM Biotin-16-UTP (Boehringher-Mannheim), 0.03% Tween-20, 2Units per μl of T7 RNA polymerase. The reactions are incubated for 90minutes at 37° C.

4. One-tenth volume of 5 M NaCl is added to the each solution containingthe biotinylated RNA generated by T7 RNA polymerase, and the resultingsolution is mixed with an equal volume of ExpressHyb reagent (Clontechlaboratories, Palo Alto, Calif.). Hybridization is performed bycontacting the amplified RNA solution, under a cover slip, with thesurface of a glass slide (Guo et al. (1994)) containing a 2.5 mm dotwith 2×10¹¹ molecules of a covalently bound 29-mer address probe withthe sequence 5'-TTTTTTTTTTTCCAAATTCTCCTCCATCA-3' (SEQ ID NO:13). Thelast 14 nucleotides of this sequence are complementary to the amplifiedmutant gene RNA, and hence the mutant RNA binds specifically. Another2.5 mm dot on the slide surface contains 2×10¹¹ molecules of acovalently bound 29-mer address probe with the sequence5'-TTTTTTTTTTTCCAAATTCTCCTCGATCA-3' (SEQ ID NO:14). The last 14nucleotides of this sequence are complementary to the amplified wildtype gene RNA, and hence the wild type RNA binds specifically. The glassslide is washed once with 2×SSPE as described (Guo et al. (1994)), thenwashed twice with 2×SSC (0.36 M sodium saline citrate), and thenincubated with fluoresceinated avidin (5 μg/ml) in 2×SSC for 20 minutesat 30° C. The slide is washed 3 times with 2×SSC and the slide-boundfluorescence is imaged at 530 nm using a Molecular Dynamics Fluorimager.

Example 4 Reverse Transcription of Ornithine Transcarbamylase (OTC) mRNAFollowed by Mutant cDNA Detection Using Gap-Filling LM-RCT

This example describes detection of human mRNA containing a mutant form(G to C) at position 114 of exon 9 of the ornithine transcarbamylasegene (Hata et al. (1988)) using cDNA generated by reverse transcription.RNA for the assay is prepared by TRIzol (Life Technologies, Inc.,Gaithersburg, Md.) extraction from liver biopsy.

1. OTC exon 9 cDNA is generated as follows:

A liver biopsy sample is stored at -80° C. in a 0.5 ml. reaction tubecontaining 40 Units of RNase inhibitor (Boehringher Mannheim). Total RNAis extracted from the frozen sample using TRIzol reagent (LifeTechnologies, Inc., Gaithersburg, Md.), and dissolved in 10 μl water. A19 μl reaction mixture is prepared containing 4 μl of 25 mM MgCl₂, 2 μlof 400 mM KCl, 100 mM Tris-HCl (pH 8.3), 8 μl of a 2.5 mM mixture ofdNTP's (dATP, dGTP, dTTP, dCTP), 1 μl of MuLV reverse transcriptase (50U, Life Technologies, Inc., Gaithersburg, Md.), 1 μl of MuLV reversetranscriptase primer (5'-TGTCCACTTTCTGTTTTCTGCCTC-3', SEQ ID NO:15), 2μl of water, and 1 μl of RNase inhibitor (20 U). The reaction mixture isadded to 1 μl of the Trizol-purified RNA solution, and incubated at 42°C. for 20 minutes to generate cDNA.

2. Two 20 μl cDNA samples from step 1 are heat-denatured for 4 minutesat 98° C., and incubated under ligation conditions in the presence oftwo 5'-phosphorylated probes:

Open circle probe (95 nucleotides):

5'-ATCACTAGTGTTCCTTCTCATAAGACTCGTCATGTCTCAGCAGCTTCTAACGGTCACTAATACGACTCACTATAGGGGATGATGAAGTCTTTT AT-3' (SEQ ID NO:16)

Gap probe for mutant gene (8 nucleotides):

5'-TAGTGATG-3'

Gap probe for wild type gene (8 nucleotides):

5'-TAGTGATC-3'

T4 DNA ligase (New England Biolabs) is added at a concentration of 5units per μl, in a buffer consisting of 10 mM Tris-HCl (pH 7.5), 0.20 MNaCl, 10 mM MgCl₂, 2 mM ATP. The concentration of open circle probe is80 nM, and the concentration of gap oligonucleotide is 100 nM. The totalvolume is 40 μliters. Ligation is carried out for 25 minutes at 37° C.

3. 25 μl are taken from each of the above reactions and mixed with anequal volume of a buffer consisting of 50 mM Tris-HCl (pH 7.5), 10 mMMgCl₂, 1 mM DTT, 200 μM each of dTTP, dATP, dGTP, dCTP; and containingthe 18-base rolling circle replication primer 5'-GCTGAGACATGACGAGTC-3'(SEQ ID NO:6), at a concentration of 0.2 μM. The φ29 DNA polymerase (160ng per 50 μl) is added and the reaction mixtures are incubated for 30minutes at 30° C.

4. To the above solutions are added compensating buffer to achieve thefollowing concentrations of reagents: 35 mM Tris-HCl (pH 8.2), 2 mMspermidine, 18 mM MgCl₂, 5 mM GMP, 1 mM of ATP, CTP, GTP, 333 μM UTP,667 μM Biotin-16-UTP (Boehringher-Mannheim), 0.03% Tween-20, 2 Units perμl of T7 RNA polymerase. The reaction is incubated for 90 minutes at 37°C.

5. One-tenth volume of 5 M NaCl is added to the each solution containingthe biotinylated RNA generated by T7 RNA polymerase, and the resultingsolution is mixed with an equal volume of ExpressHyb reagent (Clontechlaboratories, Palo Alto, Calif.). Hybridization is performed bycontacting the amplified RNA solution, under a cover slip, with thesurface of a glass slide (Guo et al. (1994)) containing a 2.5 mm dotwith 2×10¹¹ molecules of a covalently bound 29-mer address probe withthe sequence 5'-TTTTTTTTTTTTTTTTGATGGAGGAGAAT-3' (SEQ ID NO:17). Thelast 14 nucleotides of this sequence are complementary to the amplifiedmutant gene RNA, and hence the mutant RNA binds specifically. Another2.5 mm dot on the slide surface contains 2×10¹¹ molecules of acovalently bound 29-mer address probe with the sequence5'-TTTTTTTTTTTTTTTTGATCGAGGAGAAT-3' (SEQ ID NO:9). The last 14nucleotides of this sequence are complementary to the amplified wildtype gene RNA, and hence the wild type RNA binds specifically. The glassslide is washed once with 2×SSPE as described (Guo et al. (1994)), thenwashed twice with 2×SSC (0.36 M sodium saline citrate), and thenincubated with fluoresceinated avidin (5 μg/ml) in 2×SSC for 20 minutesat 30° C. The slide is washed 3 times with 2×SSC and the slide-boundfluorescence is imaged at 530 nm using a Molecular Dynamics Fluorimager.

Example 5 Multiplex Immunoassay Coupled to Rolling Circle Amplification

This example describes an example of multiplex detection of differenttarget molecules using reporter antibodies. The signal that is detectedis produced by rolling circle amplification of the target sequenceportion of the reporter antibodies.

1. Three different monoclonal antibodies, each specific for a differenttarget molecule, are coupled to three different arbitrary DNA sequences(A, B, C) that serve as unique identification tags (target sequences).In this example, the three antibodies are maleimide-modified and arespecific for β-galactosidase, hTSH, and human chorionic gonadotropin(hCG). The antibodies are coupled to aminated DNA oligonucleotides, eacholigonucleotide being 50 nucleotides long, using SATA chemistry asdescribed by Hendrickson et al. (1995). The resulting reporterantibodies are called reporter antibody A, B, and C, respectively.

2. Antibodies specific for the target molecules (not the reporterantibodies) are immobilized on microtiter dishes as follows: A 50 μlmixture containing 6 μg/ml of each of the three antibodies in sodiumbicarbonate (pH 9) is applied to the wells of a microtiter dish,incubated overnight, and washed with PBS-BLA (10 mM sodium phosphate (pH7.4), 150 mM sodium chloride, 2% BSA, 10% β-lactose, 0.02% sodium azide)to block non-adsorbed sites.

3. Serial dilutions of solutions containing one or a combination of thethree target molecules (hTSH, hCG, and β-galactosidase) are added to thewells. Some wells are exposed to one target molecule, a mixture of twotarget molecules, or a mixture of all three target molecules. After 1hour of incubation, the wells are washed three times with TBS/Tween washbuffer as described by Hendrickson et al. (1995).

4. Fifty microliters of an appropriately diluted mixture of the threereporter antibodies (A+B+C) are added to each well of the microtiterdish. The plate is incubated at 37° C. for 1 hour, and then washed fourtimes with TBS/Tween buffer.

5. To each well is added a mixture of three pairs of open circle probesand gap oligonucleotides, each pair specific for one of the three targetsequence portions of the reporter antibodies. In this example, the opencircle probes have the same spacer region of 49 bases including auniversal primer complement portion, and different 18 nucleotide targetprobe portions at each end. Each cognate pair of open circle probe andgap oligonucleotide is designed to hybridize to a specific targetsequence (A, B, or C) in the target sequence portion of the reporterantibodies. Specifically, Open circle probe A' has left and right targetprobe portions complementary to two 18-base sequences in tag sequence Aseparated by 8 bases that are complementary to the 8-nucleotide gapoligonucleotide A'. The same is the case for open circle probe and gapoligonucleotide pairs B' and C'. The concentration of each open circleprobe is 80 nM, and the concentration of each gap oligonucleotide is 120nM.

6. T4 DNA ligase (New England Biolabs) is added to each microtiter wellat a concentration of 5 units per μl, in a reaction buffer consisting(10 mM Tris-HCl (pH 7.5), 40 mM potassium acetate, 10 mM MgCl₂, 2 mMATP). The total volume in each well is 40 μliters. Ligation is carriedout for 45 minutes at 37° C.

7. To each microtiter well is added 20 μl of a compensating solutioncontaining dTTP, dATP, dGTP, dCTP (400 μM each), the universal 18-baseoligonucleotide primer 5'-GCTGAGACATGACGAGTC-3' (SEQ ID NO:6) (at afinal concentration of 0.2 μM), and φ29 DNA polymerase (at 160 ng per 50μl). The reaction for 30 minutes at 30° C.

8. After incubation, a compensating buffer is added to each well toachieve the following concentrations of reagents: 35 mM Tris-HCl (pH8.2), 2 mM spermidine, 18 mM MgCl₂, 5 mM GMP, 1 mM of ATP, CTP, GTP, 333μM UTP, 667 μM Biotin-16-UTP (Boehringher-Mannheim), 0.03% Tween-20, 2Units per μl of T7 RNA polymerase. The reaction is incubated for 90minutes at 37° C., generating biotinylated RNA.

9. One-tenth volume of 5 M NaCl is added to each well, and the resultingsolution is mixed with and equal volume of ExpressHyb reagent (Clontechlaboratories, Palo Alto, Calif.). Hybridization is performed bycontacting the mixture of amplified RNAs, under a cover slip, with thesurface of a glass slide containing three separate dots of 2×10¹¹molecules of three different covalently bound 31-mer oligonucleotides(A, B, C) (Guo et al. (1994)). The last 16 bases of each oligonucleotideare complementary to a specific segment (4 bases+8 bases+4 bases),centered on the 8-base gap sequence, of each of the possible amplifiedRNAs generated from tag sequences A, B, or C. Hybridization is carriedout for 90 minutes at 37° C. The glass slide is washed once with 2×SSPEas described (Guo et al. (1994)), then washed twice with 2×SSC (0.36 Msodium saline citrate), and then incubated with fluoresceinated avidin(5 μg/ml) in 2×SSC for 20 minutes at 30° C. The slide is washed 3 timeswith 2×SSC and the surface-bound fluorescence is imaged at 530 nm usinga Molecular Dynamics Fluorimager to determine if any of tag sequences Aor B or C was amplified.

Example 6 In situ Detection of Ornithine Transcarbamylase (OTC) andCystic Fibrosis (CF) Target Sequences Using LM-RCA

1. DNA samples were prepared as follows:

A sample of lymphocytes was washed twice in PBS, with the cellscollected by centrifugation for 5 minutes at 1500 RPM. The cells wereresuspended in 10 mM PIPES, pH 7.6, 100 mM NaCl, 0.3 M sucrose, 3 mMMgCl₂, and 0.5% Triton X-100. The cells were then incubated on ice for15 minutes, centrifuged for 5 minutes at 1700 RPM, and resuspend at2×10⁵ nuclei/ml. Samples of 1.0×10⁵ nuclei (0.5 ml) were centrifugedonto slides (5 minutes at 500 g, setting #85) in Cytospin centrifuge.The slides were then rinsed twice for 3 minutes with PBS, rinsed oncefor 6 minutes with agitation in 2 M NaCl, 10 mM PIPES, pH 6.8, 10 mMEDTA, 0.5% Triton X-100, 0.05 mM Spermine, and 0.125 mM Spermidine. Theslides were then rinsed for one minute in 10×PBS, for one minute in5×PBS, for one minute in 2×PBS, for 2 minutes in 1×PBS, for one minutein 10% ethanol, for one minute in 30% ethanol, for one minute in 70%ethanol, and for one minute in 95% ethanol. Finally, the slides were airdried and then fixed by baking at 70° C. for 2 hours.

2. The following DNA molecules were used:

OTC Open Circle Probe (OTC OCP, for OTC target sequence):

5'-GAGGAGAATAAAAGTTTCTCATAAGACTCGTCATGTCTCAGCAGCTTCTAACGGTCACTAATACGACTCACTATAGGTTCTGCCT CTGGGAACAC-3'

OTC Gap oligonucleotide:

5'-TAGTGATC-3'

Cystic fibrosis Open Circle Probe (CF OCP, for CF target sequence):

5'-TATTTTCTTTAATGGTTTCTCTGACTCGTCATGTCTCAGCTTTAGTTTAATACGACTCACTATAGGATCTATATTCATCAT AGGAAACAC-3'

Cystic fibrosis Gap oligonucleotide

5'-CAAAGATGA-3'

3. DNA on the sample slides was denatured by washing the slides for 5minutes in 2×SSC, incubating in denaturation buffer (2×SSC, 70%formamide, pH 7.2) for 1 minute and 45 seconds in a pre-heated largeCoplin jar at 71° C. Heating was stopped immediately by washing theslides for three minutes in ice-cold 70% ethanol, for two minutes in 90%ethanol, and for three minutes in 100% ethanol.

4. LM-RCA was performed as follows:

In three separate reactions, the OCPs and gap oligonucleotides werehybridized and ligated to target sequences on the sample slides.

a. OTC and CF ligation operation: 42 μl of the mixture below was placedon each of two slides.

    ______________________________________                                         9 μl                                                                              10X ligation buffer (Ampligase)                                          5 μl BSA, 2 mg/ml stock                                                    9 μl OTC Gap oligo (15 μM) [final 1500 nM]                              9 μl CF Gap oligo (10 μM) [final 1000 nM]                               3 μl OTC OCP, (6 μM stock) [final = 200 nMolar]                         3 μl CF OCP, (6 μM stock) [final = 200 nMolar]                         15 μl Ampligase (5 U/μl) [final = 0.833 U/μl]                        38 μl H.sub.2 O                                                          ______________________________________                                    

The reaction was incubated for 120 minutes at 50° C.

b. OTC ligation operation: 42 μl of the mixture below was placed on aslide.

    ______________________________________                                          6 μl                                                                             10X ligation buffer (Ampligase)                                         3.5 μl BSA, 2 mg/ml stock                                                    6 μl OTC Gap oligo (15 μM) [final 1500 nM]                              2 μl OTC OCP, (6 μM stock) [final = 200 nMolar]                        10 μl Ampligase (5 U/μl) [final = 0.833 U/μl]                        33 μl H.sub.2 O                                                         ______________________________________                                    

The reaction was incubated for 120 minutes at 50° C.

c. CF ligation operation: 42 μl of the mixture below was placed on aslide.

    ______________________________________                                          6 μl                                                                             10X ligation buffer (Ampligase)                                         3.5 μl BSA, 2 mg/ml stock                                                    6 μl CF Gap oligo (10 μM) [final 1000 nM]                               2 μl CF OCP, (6 μM stock) [final = 200 nMolar]                         10 μl Ampligase (5 U/μl) [final = 0.833 U/μl]                        33 μl H.sub.2 O                                                         ______________________________________                                    

The reaction was incubated for 120 minutes at 50° C.

All of the slides were washed twice for 5 minutes with 2×SSC with 20%formamide at 42° C., washed for two minutes with 20 mM Tris, pH 7.5,0.075 M NaCl to remove the formamide, and washed for three minutes with50 mM Tris, pH 7.5, 40 mM KOAc, 10 mM MgCl₂, 10 mM DTT, 100 μg/ml BSA.

The amplification operation was performed by placing 24 μl of thefollowing mixture on each slide.

    ______________________________________                                        18.0 μl                                                                             H.sub.2 O [total volume = 100 μl for 4 slides]                      20.0 μl 5X φ29 buffer with BSA BSA is 200 μg/ml                     16.0 μl dNTPs (A, G, and C, each 2.5 mM)                                    5.0 μl dTTP (2.5 mM)                                                      15.0 μl BUdR (2.5 mM)                                                       7.0 μl rolling circle replication primer (10 μM)                        3.0 μl Gene32 Protein (1.37 μg/μl) (final 41 μg/ml)                      16.0 μl φ29 DNA polymerase (1:6 dilution, 16 μl = 768              ng)                                                                  ______________________________________                                    

The reaction was incubated 20 minutes in 37° C. oven.

All slides were then washed twice for four minutes with 2×SSC with 20%formamide at 25° C., and then washed twice for four minutes with 2×SSC,3% BSA, 0.1% Tween-20 at 37° C.

5. The TS-DNA generated in the amplification operation was collapsed anddetected as follows:

50 μl of a solution of AntiBUDR-Mouse.IgG (7 μg/ml) in 2×SSC, 3% BSA,0.1% Tween-20 was placed on each slide, and the slides were incubatedfor 30 minutes at 37° C. Then the slides were washed three times forfive minutes with 2×SSC, 3% BSA, 0.1% Tween-20 at 37° C. Next, 50 μl ofa solution of FITC-Avidin (6 μg/ml) was placed on each slide, and theslides were incubated for 30 minutes at 37° C. Then the slides werewashed three times for five minutes with 2×SSC, 3% BSA, 0.1% Tween-20 at37° C., and then incubated for 2.6 minutes with 2×SSC, 0.1 μg/ml DAPI(26 μl in 50 ml) at room temp. Next, the slides were washed 10 minuteswith 1×SSC, 0.01% Tween at room temperature and then covered with 24 μlantifade. Finally, the slides were examined in a microscope with CCDcamera for DAPI nuclear fluorescence and discrete fluorescein signals.

Example 7 Multiplex Detection of Multiple Target Sequences UsingLM-RCA-CMC

This example illustrates multiplex detection using 31 different OCPs andgap oligonucleotide pairs, each designed to generate 31 different colorcombinations using 5 basic colors.

1. Slides containing samples are prepared as follows:

Poly-L-Lysine coated microscope slides are prepared, and DNA is spottedusing an arraying machine as described above using the method describedby Schena et al. The size of each spot of sample DNA is 2.5 mm. DNA isdenatured as described above using the method described by Schena et al.

2. A mixture of gap oligonucleotides and open circle probes is designedand prepared, containing 31 different OCPs and 31 different gapoligonucleotides. The OCPs and gap oligonucleotides are designed aspairs with each OCP and gap probe pair containing sequencescomplementary to a specific target sequence of interest. The spacerregions of each of the 31 OCPs contain unique, alternative combinationsof five possible detection tags, designated 1t, 2t, 3t, 4t, and 5t. Thecombinations are coded according to the scheme shown below. The set ofpairs is designated as follows:

    ______________________________________                                        Gap oligo   OCP     1t      2t  3t    4t  5t                                  ______________________________________                                        g1          ocp1    +                                                           g2 ocp2  +                                                                    g3 ocp3   +                                                                   g4 ocp4    +                                                                  g5 ocp5     +                                                                 g6 ocp6 + +                                                                   . . .                                                                         . . . and so on                                                               . . .                                                                         g25 ocp25   + + +                                                             g26 ocp26 + + + +                                                             g27 ocp27 + + +  +                                                            g28 ocp28 + +  + +                                                            g29 ocp29 +  + + +                                                            g30 ocp30  + + + +                                                            g31 ocp31 + + + + +                                                         ______________________________________                                    

3. LM-RCA is performed as follows:

The OCPs and gap oligonucleotides are hybridized and ligated to targetsequences on the sample slides with 50 μl of the following mixture.

    ______________________________________                                        1.5 μl                                                                            10X ligation buffer (Ampligase)                                          8.8 μl BSA, 2 mg/ml stock                                                   15 μl Mixture of 31 Gap oligonucleotides [final 400 nM for each]                  5 μl Mixture of 31 OCPs [final = 100 nMolar for each]                     25 μl Ampligase (5 U/μl)                                          82 μl H.sub.2 O                                                         ______________________________________                                    

The reaction is incubated for 60 minutes at 52° C.

The slides are washed twice for 5 minutes with 2×SSC with 20% formamideat 42° C., washed for two minutes with 20 mM Tris, pH 7.5, 0.075 M NaClto remove the formamide, and washed for three minutes with 50 mM Tris,pH 7.5, 40 mM KOAc, 10 mM MgCl₂, 10 mM DTT, 100 μg/ml BSA.

The amplification operation is performed by placing 24 μl of thefollowing mixture on each slide.

    ______________________________________                                        18.0 μl                                                                             H.sub.2 O [total volume = 100 μl for 4 slides]                      20.0 μl 5X φ29 buffer with BSA BSA is 200 μg/ml                     16.0 μl dNTPs (A, G, and C, each 2.5 mM)                                    5.0 μl dTTP (2.5 mM)                                                      15.0 μl BUdR (2.5 mM)                                                       7.0 μl rolling circle replication primer (10 μM)                        3.0 μl Gene32 Protein (1.37 μg/μl) (final 41 μg/ml)                      16.0 μl φ29 DNA polymerase (1:6 dilution, 16 μl = 768              ng)                                                                  ______________________________________                                    

The reaction is incubated 15 minutes in 37° C. oven.

All slides were then washed twice for four minutes with 2×SSC with 20%formamide at 25° C.

4. The 5 collapsing detection probes, each with a different label andeach complementary to one of the 5 detection tags, are hybridized to theTS-DNA on the slides in a solution of 4×SSC. The detection probescorrespond to the detection tags as follows:

    ______________________________________                                        Detection probe                                                                              Label     Detection tag                                        ______________________________________                                        dp1            fluorescein                                                                             1t                                                     dp2 Cy3 2t                                                                    dp3 Cy3.5 3t                                                                  dp4 Cy5 4t                                                                    dp5 Cy7 5t                                                                  ______________________________________                                    

All slides were then washed twice for four minutes with 2×SSC with 20%formamide at 25° C., and then washed twice for four minutes with 2×SSC,3% BSA, 0.1% Tween-20 at 37° C.

5. The TS-DNA generated in the amplification operation is furthercollapsed and detected as follows:

50 μl of a solution of AntiBUDR-Mouse.IgG (7 μg/ml) in 2×SSC, 3% BSA,0.1% Tween-20 is placed on each slide, and the slides are incubated for30 minutes at 37° C. Then the slides are washed three times for fiveminutes with 2×SSC, 3% BSA, 0.1% Tween-20 at 37° C. Next, 50 μl of asolution of Avidin DN (6 μg/ml) in 2×SSC, 3% BSA, 0. 1% Tween-20 isplaced on each slide, and the slides are incubated for 30 minutes at 37°C. Then the slides are washed three times for five minutes with 2×SSC,3% BSA, 0.1% Tween-20 at 37° C., washed 5 minutes with 2×SSC, 0.01%Tween at room temperature, and then covered with 24 μl antifade.Finally, the slides are scanned in a fluorescence scanning device withappropriate filters (for example, those described by Schena et al.).Image analysis software is used to count and analyze the spectralsignatures of the fluorescent dots.

Example 8 Multiplex Detection of Multiple Target Sequences UsingLM-RCA-CMC

This example illustrates multiplex detection using 15 different OCPs and30 different gap oligonucleotides, where pairs of gap oligonucleotidesare associated with each OCP. The OCPs and gap oligonucleotides aredesigned to generate 30 different color combinations using 6 basic labelcolors.

1. Slides containing samples are prepared as follows:

Poly-L-Lysine coated microscope slides are prepared, and DNA is spottedusing an arraying machine as described above using the method describedby Schena et al. The size of each spot of sample DNA is 2.5 mm. DNA isdenatured as described above using the method described by Schena et al.

2. A mixture of gap oligonucleotides and open circle probes is designedand prepared, containing 15 different OCPs and 30 different gapoligonucleotides. The OCPs and gap oligonucleotides are designed aspairs with each OCP and gap probe pair containing sequencescomplementary to a specific target sequence of interest. The spacerregions of each of the 15 OCPs contain unique, alternative combinationsof four possible detection tags, designated 1t, 2t, 3t, and 4t.Additional detection tags are generated by ligation of an OCP to a gapoligonucleotide. These form two different detection tags depending onwhich of the pair of gap oligonucleotides is ligated to a given OCP. Thecombinations are coded according to the scheme shown below. The set ofpairs is designated as follows:

    ______________________________________                                        Gap oligo   OCP       1t    2t      3t  4t                                    ______________________________________                                        g1          ocp1      +                                                         g2 ocp1 +                                                                     g3 ocp2  +                                                                    g4 ocp2  +                                                                    g5 ocp3   +                                                                   g6 ocp3   +                                                                   . . .                                                                         . . . and so on                                                               . . .                                                                         g25 ocp13 +  + +                                                              g26 ocp13 +  + +                                                              g27 ocp14  + + +                                                              g28 ocp14  + + +                                                              g29 ocp15 + + + +                                                             g30 ocp15 + + + +                                                           ______________________________________                                    

3. LM-RCA is performed as follows:

The OCPs and gap oligonucleotides are hybridized and ligated to targetsequences on the sample slides with 50 μl of the following mixture.

    ______________________________________                                        1.5 μl                                                                            10X ligation buffer (Ampligase)                                          8.8 μl BSA, 2 mg/ml stock                                                   15 μl Mixture of 30 Gap oligonucleotides [final 400 nM for each]                  5 μl Mixture of 15 OCPs [final = 100 nMolar for each]                     25 μl Ampligase (5 U/μl)                                          82 μl H.sub.2 O                                                         ______________________________________                                    

The reaction is incubated for 60 minutes at 52° C.

The slides are washed twice for 5 minutes with 2×SSC with 20% formamideat 42° C., washed for two minutes with 20 mM Tris, pH 7.5, 0.075 M NaClto remove the formamide, and washed for three minutes with 50 mM Tris,pH 7.5, 40 mM KOAc, 10 mM MgCl₂, 10 mM DTT, 100 μg/ml BSA.

The amplification operation is performed by placing 24 μl of thefollowing mixture on each slide.

    ______________________________________                                        18.0 μl                                                                             H.sub.2 O [total volume = 100 μl for 4 slides]                      20.0 μl 5X φ29 buffer with BSA BSA is 200 μg/ml                     16.0 μl dNTPs (A, G, and C, each 2.5 mM)                                    5.0 μl dTTP (2.5 mM)                                                      15.0 μl BUdR (2.5 mM)                                                       7.0 μl rolling circle replication primer (10 μM)                        3.0 μl Gene32 Protein (1.37 μg/μl) (final 41 μg/ml)                      16.0 μl φ29 DNA polymerase (1:6 dilution, 16 μl = 768              ng)                                                                  ______________________________________                                    

The reaction is incubated 15 minutes in 37° C. oven.

All slides were then washed twice for four minutes with 2×SSC with 20%formamide at 25° C.

4. Four collapsing detection probes, each with a different label andeach complementary to one of the 4 detection tags, 1t, 2t, 3t, and 4t,along with 30 collapsing detection probes, each with one of two labelsand each complementary to one of the detection tags formed by theligation of an OCP and gap oligonucleotide, are hybridized to the TS-DNAon the slides in a solution of 4×SSC. The detection probes correspond tothe detection tags as follows:

    ______________________________________                                        Detection probe                                                                              Label     Detection tag                                        ______________________________________                                        dp1            fluorescein                                                                             1t                                                     dp2 Cy3 2t                                                                    dp3 Cy3.5 3t                                                                  dp4 Cy5.5 4t                                                                  dp5 Cy5 g1                                                                    dp6 Cy7 g2                                                                    dp7 Cy5 g3                                                                    dp8 Cy7 g4                                                                    dp9 Cy5 g5                                                                    dp10 Cy7 g6                                                                   dp11 Cy5 g7                                                                   dp12 Cy7 g8                                                                   dp13 Cy5 g9                                                                   dp14 Cy7 g10                                                                  dp15 Cy5 g11                                                                  dp16 Cy7 g12                                                                  dp17 Cy5 g13                                                                  dp18 Cy7 g14                                                                  dp19 Cy5 g15                                                                  dp20 Cy7 g16                                                                  dp21 Cy5 g17                                                                  dp22 Cy7 g18                                                                  dp23 Cy5 g19                                                                  dp24 Cy7 g20                                                                  dp25 Cy5 g21                                                                  dp26 Cy7 g22                                                                  dp27 Cy5 g23                                                                  dp28 Cy7 g24                                                                  dp29 Cy5 g25                                                                  dp30 Cy7 g26                                                                  dp31 Cy5 g27                                                                  dp32 Cy7 g28                                                                  dp33 Cy5 g29                                                                  dp34 Cy7 g30                                                                ______________________________________                                    

All slides were then washed twice for four minutes with 2×SSC with 20%formamide at 25° C., and then washed twice for four minutes with 2×SSC,3% BSA, 0.1% Tween-20 at 37° C.

5. The TS-DNA generated in the amplification operation is furthercollapsed and detected as follows:

50 μl of a solution of AntiBUDR-Mouse.IgG (7 μg/ml) in 2×SSC, 3% BSA,0.1% Tween-20 is placed on each slide, and the slides are incubated for30 minutes at 37° C. Then the slides are washed three times for fiveminutes with 2×SSC, 3% BSA, 0.1% Tween-20 at 37° C. Next, 50 μl of asolution of Avidin DN (6 μg/ml) in 2×SSC, 3% BSA, 0.1% Tween-20 isplaced on each slide, and the slides are incubated for 30 minutes at 37°C. Then the slides are washed three times for five minutes with 2×SSC,3% BSA, 0.1% Tween-20 at 37° C., washed 5 minutes with 2×SSC, 0.01%Tween at room temperature, and then covered with 24 μl antifade.Finally, the slides are scanned in a fluorescence scanning device withappropriate filters (for example, those described by Schena et al.).Image analysis software is used to count and analyze the spectralsignatures of the fluorescent dots.

Example 9 Unimolecular Segment Amplification and Sequencing

This example illustrates unimolecular segment amplification (that is,rolling circle amplification) followed by single nucleotide primerextension sequencing. In this example, an OCP is hybridized to a targetnucleic acid so as to leave a gap in a region of known sequencevariation. After formation of an amplification target circle usinggap-filling ligation, and rolling circle amplification of theamplification target circle, the amplified DNA is subjected to chainterminating primer extension sequencing using uniquely labeledchain-terminating nucleotides. Detection of the incorporated labelidentifies the nucleotide of interest.

An Open Circle Probe designed to hybridize with the Cystic FibrosisTransmembrane Regulator G542X mutant locus is designed so as to leave agap of four bases, encompassing the mutant base. The gap is to be filledby a DNA polymerase in a gap-filling ligation operation, therebyincorporating whatever sequence is present in the target Cystic FibrosisTransmembrane Regulator G542X.

The sequence of the 5'-phosphorylated OCP (82 bases) is as follows:

GAACTATATTGTCTTTCTCTGTTTTCTTGCATGGTCACACGTCGTTCTAGTACGCTTCTAACTTAGTGTGATTCCACCTTCT (nucleotides 1 to 82 of SEQ ID NO:20)

The underlined ends of this probe hybridize with the target human DNA asindicated below (target sequence shown in reverse, 3' to 5'): (mutant)(t) gtgagtcacactaaggtggaagaggttcttgatataacagaaagagacgtttga ||||||.vertline.|||.vertlin e.|||.vertline .|||| ||||. vertline.|||.v ertline.|||.vertline.|||.ver tline.|||.vert line.||| AGTGTGATTCCACCTTCT GAACTATATTGTCTTTCTCTG Left target probe Right target probe 18 gap 21

The target DNA is SEQ ID NO:21. The left target probe is nucleotides 65to 82 of SEQ ID NO:20. The right target probe is nucleotides 1 to 21 ofSEQ ID NO:20. The region in the target DNA opposite the gap encompassesa nucleotide which is either g (wild type) or t (mutant). It is thisnucleotide position which is interrogated (that is, sequenced).

1. Microscope slides containing bound DNA samples are prepared asdescribed by Schena et al.

2. Gap-filling ligation in the presence of 150 nMolar Open Circle Probe,Ampligase DNA ligase, and Thermus flavus DNA polymerase is carried outas described earlier (generally using the conditions described byAbravaya et al., Nucleic Acids Research 23:675-682 (1995)), in thepresence of dATP and dCTP, so that the gap is filled and immediatelyligated. The reaction is carried out with the slides covered with a 22by 40 mm cover slip, in a volume of 28 μl, and is incubated for 1 hourat 58° C. The filling reaction adds the base sequence CCAA for the wildtype, or the sequence CAAA for the mutant gene, respectively.

3. Wash slides twice in 2×SSC with 20% formamide for 5 minutes at 42° C.

4. Wash slides for 2 minutes with 20 mM Tris, pH 7.5, 0.075 M NaCl.

5. Rolling Circle Amplification is carried out in situ for 15 minutes at30° C. in a buffer containing the following components: 50 mM Tris-HCl,pH 7.5, 10 mM MgCl₂, 1 mM DTT, 400 μM each of dCTP, dATP, and dGTP, 95μM dTTP, 380 μM BUDR triphosphate (SIGMA), the 18 nucleotide rollingcircle replication primer, ACGACGTGTGACCATGCA (SEQ ID NO:22), at aconcentration of 0.7 μM, Phage T4 Gene-32 protein at a concentration of1000 nMolar, and φ29 DNA polymerase at 200 nM. This reaction generatesapproximately 400 copies of TS-DNA containing faithful copies of thegene sequence.

6. Wash twice in 2×SSC with 20% formamide for 5 minutes at 25° C.

7. Incubate the slides with the 20 nucleotide interrogation primer,TAGTGTGATTCCACCTTCTC (nucleotides 64 to 83 of SEQ ID NO:20), designed tohybridize with the TS-DNA adjacent to the nucleotide being interrogated,shown below as a boldface N: Primer TAGTGTGATTCCACCTTCTC |||||||||.vertline .|||.vertlin e.|||.vertli ne.||TS-DNA...gaagattgaatcacactaaggtggaagagNttcttgata...

The TS-DNA is SEQ ID NO:22. The slides are incubated 10 minutes at 37°C. in the following conditions:

Four Units Sequenase DNA polymerase (Amersham-USB), in 25 μl of 50 mMTris-HCl, pH 7.5, 20 mM MgCl₂, 50 mM NaCl, 5 mM DTT, 50 μg/ml BovineSerum Albumin (Molecular Biology grade from Life Sciences, Inc.), 50 μMfluorescent-ddATP, fluorescent-ddCTP, fluorescent-ddGTP, andfluorescent-ddTTP. The four fluorescent dideoxynucleoside triphosphateseach have different emission spectra and can be obtained from a standardDNA sequencing kit (Applied Biosystems, Inc.).

Sequenase DNA polymerase incorporates only one ddNTP, where theincorporated ddNTP is complementary to the nucleotide beinginterrogated. This is illustrated below where when the interrogatednucleotide is a T (the mutant form), fluorescent ddATP is incorporated:Extended primer TAGTGTGATTCCACCTTCTCA*|||||.vertline.||||||.vertline.|||||||.vertline.TS-DNA...gaagattgaatcacactaaggtggaagagTttcttgata...

The extended primer is nucleotides 64 to 84 of SEQ ID NO:20. If theinterrogated nucleotide is G (the wild type), fluorescent ddCTP isincorporated.

8. Wash for 5 minutes in 2×SSC at 25° C.

9. Wash for 4 minutes in 2×SSC, 2.8% BSA, 0.12% Tween-20 at 37° C.

10. Incubate 30 minutes at 37° C. in 50 μl (under cover slip) using 5μg/ml Biotinylated AntiBUDR-Mouse.IgG (Zymed Labs) in 2×SSC, 2.8% BSA,and 0.12% Tween-20.

11. Wash three times in 2×SSC, 2.8% BSA, and 0.12% Tween-20 for 5minutes at 37° C.

12. Incubate 30 minutes at 37° C. in 50 μl (under cover slip) usingFITC-Avidin, 5 μg/ml, in 2×SSC, 2.8% BSA, and 0.12% Tween-20.

13. Wash three times in 2×SSC, 2.8% BSA, and 0.12% Tween-20 for 5minutes at 37° C.

14. Wash 5 minutes with 2×SSC, and 0.01% Tween-20 at room temperature.

15. An image of the slide is captured using a microscope-CCD camerasystem with appropriate filter sets. Each TS-DNA, each with multipleextended primers, occupy a small area on the slides. The incorporatedfluorescent nucleotides produce individual fluorescent dots for eachTS-DNA. The fluorescent emission color defines the nucleotideincorporated at the specific extension position in each fluorescent dot.Thus, in a sample containing a mixture of wild type and mutantsequences, the presence of each is indicated by the presence offluorescent dots having the fluorescent emission color of thefluorescent ddATP (indicating the mutant form) and of fluorescent dotshaving the fluorescent emission color of the fluorescent ddCTP(indicating the wild type). The dots will be distinct anddistinguishable due to the small area occupied by each TS-DNA due to itscollapse.

Expected results for heterozygous and homozygous samples are depicted inFIG. 16. The large circles represent a target sample dot on the slide.The small circles represent individual TS-DNA molecules, amplified insitu at the location of target nucleic acids in the sample, which havebeen subjected to primer extension sequencing. In an actual assay,hundreds or thousands of individually detectable TS-DNA molecules wouldbe present in a sample dot, and the area occupied by the collapsedTS-DNA would be much smaller. Fewer and larger TS-DNA spots are depictedin FIG. 16 for clarity of illustration. The nucleotide incorporated isidentified by its fluorescent spectrum and is based on the nucleotidepresent at the interrogated position in the TS-DNA. FIG. 16A isrepresentative of a sample that is homozygous for the wild type sequence(indicated by incorporation of cystine). All of the cells in the sample(and thus, all of the target nucleic acids in the sample) have the samesequence resulting in the same incorporated nucleotide for all of theTS-DNA molecules in the sample. FIG. 16B is representative of a samplethat is heterozygous for the wild type and a mutant (indicated by anequal number of TS-DNA molecules resulting in incorporation of cystineand adenine). All of the cells in the sample (and thus, all of thetarget nucleic acids in the sample) have one copy of both sequences(that is, wild type and mutant), resulting in the incorporation of twodifferent nucleotides; each for half of the TS-DNA molecules in thesample. FIG. 16C is representative of a sample that is homozygous butincludes a few cells with a somatic mutation. Most of the cells in thesample (and thus, most of the target nucleic acids in the sample) havethe same sequence (that is, wild type), and only a few have the mutantsequence. This results in the incorporation of one nucleotide for mostof the TS-DNA molecules, and incorporation of a different nucleotide fora few TS-DNA molecules. The ratio of the number of TS-DNA molecules forwhich a given nucleotide is incorporated is an accurate measure of theratio of the corresponding target nucleic acid in the sample. Suchsensitive detection of somatic mutations will be particularly useful fordetecting, for example, a few cancer cells, or a few virally infectedcells, in a sample containing mostly normal or uninfected cells.

Example 10 Unimolecular Segment Amplification and CAGE Sequencing

This example illustrates unimolecular segment amplification (that is,rolling circle amplification) followed by degenerate probe primerextension sequencing using caged oligonucleotides. In this example, anOCP is hybridized to a target nucleic acid so as to leave a gap in aregion of known sequence variation. After formation of an amplificationtarget circle using gap-filling ligation, and after rolling circleamplification of the amplification target circle, interrogation probesare hybridized to the amplified DNA. Interrogation primers are thenformed by ligating degenerate probe to the interrogation probes. Theinterrogation primers are then extended in chain terminating primerextension sequencing using uniquely labeled chain-terminatingnucleotides. This example illustrates the use of sequential addition ofdegenerate probes to hybridized interrogation probes in an arrayedsolid-state sample. Detection of the incorporated label identifies thenucleotide sequence in the region of interest.

An Open Circle Probe (OCP.96) of 96 bases with a 5'-phosphate isdesigned to hybridize with a GT repeat polymorphic locus. The probe isdesigned to leave a gap in the GT repeat region when hybridized to thetarget DNA. The gap is to be filled by a DNA polymerase in a gap-fillingligation operation, thereby incorporating the entire GT repeat regioninto the ligated OCP.

The sequences of the OCP (96 bases) is as follows:

ATCTAGCTATGTACGTACGTGAACTTTTCTTGCATGGTCACACGTCGTTCTAGTACGCTTCTAACTTTTAACATATCTCGACATCTAACGATCG AT (nucleotides 1 to 96of SEQ ID NO:25)

The underlined ends of this probe hybridize with the target DNA asindicated in FIG. 17. In FIG. 17, the gap space is indicated as "Fillsequence". FIG. 17A shows hybridization of the OCP to target DNA having10 repeats of CA. FIG. 17B shows hybridization of the OCP to target DNAhaving 9 repeats of CA. As will be shown, USA-CAGESEQ is a useful andaccurate method of determining the nucleotide sequence in a highlyrepetitive region of DNA.

1. Five microscope slides, each containing at least one verticallyaligned column of five identical bound denatured DNA samples areprepared as described by Schena et al. Each slide may contain from oneto 100 regularly spaced columns of DNA samples, as long as the number ofsample dots in each column is five. The slides should be identical (orat least have an identical set of DNA samples). An example of a slidewith an array of bound DNA samples is shown in FIG. 18A. The five sampledots in each column are identical (that is, they are from the same DNAsample). Each column of sample dots is preferably made from differentsample samples.

2a. The slides are incubated in 50 mM Tris-HCl, pH 7.5, 0.3 M NaCl, 0.5mM EDTA, and 150 nM OCP.96 oligo, for 1 hour at 48° C. to achievehybridization of the OCP. The slides are then washed for 2 minutes in 50mM Tris-HCl, pH 7.5, 100 mM M KCl, and 0.05% Triton X-100.

2b. Gap-filling ligation is carried out in the presence of Ampligase DNAligase and Thermus flavus DNA polymerase using the conditions describedabove (generally using conditions described by Abravaya et al.) in thepresence of dATP, dCTP, dGTP, and dTTP, so that the gap is filled andimmediately ligated. The incubation is carried out with the slidescovered with a 22 by 40 mm cover slip, in a volume of 28 μl, for 45minutes at 54° C.

3. Wash slides twice in 2×SSC with 20% formamide for 5 minutes at 42° C.

4. Wash slides for 2 minutes with 20 mM Tris, pH 7.5, and 0.075 M NaCl.

5. Rolling Circle Amplification is carried out in situ for 15 minutes at30° C. in a buffer containing the following components: 50 mM Tris-HClpH 7.5, 10 mM MgCl₂, 1 mM DTT, 400 μM each of dCTP, dATP, dGTP, 95 μMdTTP, 360 μM BUDR triphosphate (SIGMA), the 18 nucleotide rolling circlereplication primer, ACGACGTGTGACCATGCA (SEQ ID NO:22), at aconcentration of 700 nM, Phage T4 Gene-32 protein at a concentration of1000 nMolar, and φ29 DNA polymerase at 200 nM. This reaction generatesapproximately 400 copies of TS-DNA containing faithful copies of thetarget DNA.

6. Wash twice in 2×SSC with 20% formamide for 5 minutes at 25° C.

7a. Incubate one slide (slide number 1) in 2×SSC and 300 nMolar of afirst 20 nucleotide interrogation probe (interrogation probe 1),TCTCGACATCTAACGATCGA (nucleotides 76 to 95 of SEQ ID NO:25), whichhybridizes with the TS-DNA.

7b. Incubate another slide (slide number 2) in 2×SSC and 300 nMolar of asecond 20 nucleotide interrogation probe (interrogation probe 2),CTCGACATCTAACGATCGAT (nucleotides 77 to 96 of SEQ ID NO:25), whichhybridizes with the TS-DNA.

7c. Incubate another slide (slide number 3) in 2×SSC and 300 nMolar of athird 20 nucleotide interrogation probe (interrogation probe 3),TCGACATCTAACGATCGATC (nucleotides 78 to 97 of SEQ ID NO:25), whichhybridizes with the TS-DNA.

7d. Incubate another slide (slide number 4) in 2×SSC and 300 nMolar of afourth 20 nucleotide interrogation probe (interrogation probe 4),CGACATCTAACGATCGATCC (nucleotides 79 to 98 of SEQ ID NO:25), whichhybridizes with the TS-DNA.

7e. Incubate another slide (slide number 5) in 2×SSC and 300 nMolar of afifth 20 nucleotide interrogation probe (interrogation probe 5),GACATCTAACGATCGATCCT (nucleotides 80 to 99 of SEQ ID NO:25), whichhybridizes with the TS-DNA.

The five interrogation probes constitute a nested set as describedearlier. Their relationship to the amplified TS-DNA is shown below:Probe 1 TCTCGACATCTAACGATCGA Probe 2 CTCGACATCTAACGATCGAT Probe 3TCGACATCTAACGATCGATC Probe 4 CGACATCTAACGATCGATCC Probe 5GACATCTAACGATCGATCCT ||||||.vertli ne.||||||.ver tline.||||||TS-DNATAGAGCTGTAGATTGCTAGCTAGGATCACACACACACACACA

Probe 1 is nucleotides 76 to 95 of SEQ ID NO:25, probe 2 is nucleotides77 to 96 of SEQ ID NO:25, probe 3 is nucleotides 78 to 97 of SEQ IDNO:25, probe 4 is nucleotides 79 to 98 of SEQ ID NO:25, probe 5 isnucleotides 80 to 99 of SEQ ID NO:25, and the TS-DNA (shown 3' to 5') isnucleotides 19 to 60 of SEQ ID NO:19.

8. The slides are washed for 2 minutes in 50 mM Tris-Cl pH 7.5, 150 mMKCl, and 0.05% Triton X-100.

9. The slides are then subjected to five sequential rounds of degenerateprobe ligation. Each round consists of the following steps:

(a) The 5 slides are incubated with a ligation reaction mixture thatcontains the following components:

(i) A full set of pentamer degenerate probes (that is, a mixture ofoligonucleotides representing all 1,024 possible pentameric sequences),each degenerate probe at a concentration of 40 nMolar, where eachdegenerate probe has a 5' phosphate and a modified nucleotide at the 3'end (that is, a block at the 3' end). In this example, the modifiednucleotides are caged nucleotides which are of the following form:

2'-Deoxy-3'-O-(2-nitrobenzyl)adenosine

2'-Deoxy-3'-O-(2-nitrobenzyl)guanosine

2'-Deoxy-3'-O-(2-nitrobenzyl)thymidine

2'-Deoxy-3'-O-(2-nitrobenzyl)cytosine

These nucleotides are described by Metzker et al., Nucleic AcidsResearch 22:4259-4267 (1994). The modified bases protect (that is,block) the 3'-hydroxyl and render the degenerate probes incapable ofparticipating in DNA polymerase extension or DNA ligation.

(ii) A suitable DNA ligase, preferably Phage T4 DNA ligase.

Ligation is carried out with T4 DNA ligase (New England Biolabs) at aconcentration of 8 units per μl, in a buffer consisting of 10 mMTris-HCl, pH 7.5, 0.18 M NaCl, 12 mM MgCl₂, 2 mM ATP, and 10%polyethylene glycol. The total volume is 25 μl. Ligation is carried outfor 40 minutes at 32° C.

(b) A primer extension reaction is carried out for 5 minutes at 37° C.in 25 μl, under a cover slip, in the presence of 5 Units Sequenase DNApolymerase (Amersham-USB), 50 mM Tris-HCl, pH 7.5, 20 mM MgCl₂, 50 mMNaCl, 5 mM DTT, 50 μg/ml Bovine Serum Albumin (Molecular Biology gradefrom Life Sciences, Inc.) 50 μM ddATP, ddCTP, ddGTP, ddTTP. Thisreaction blocks all the primer 3' ends that failed to participate in aligation event. The slides are washed for 5 minutes in 2×SSC at 25° C.to remove any unligated degenerate probes.

After these steps, all of the interrogation probes are ligated to adegenerate probe. In the first round of degenerate probe ligation, anopaque "mask" is laid over the first row of DNA sample dots in all ofthe slides (see FIG. 18B). This mask thus covers the first sample dot ofeach column. The slides are exposed to UV light for 4 minutes to removethe cage structures from all the ligated degenerate probes in all thesample rows except row 1, which is not illuminated.

For the second round of degenerate probe ligation, steps (a) and (b) arerepeated. Degenerate probes can only be ligated to DNA sample dots inrows 2 to 5 since the cage structure remains at the 3' end of thedegenerate probes ligated to the DNA sample dots in the first row. Afterthese steps, the interrogation probes in rows 2 to 5 are ligated to twodegenerate probes. Then the opaque mask is laid over the first andsecond rows of DNA sample dots in all of the slides (see FIG. 18C). Themask thus covers the first and second sample dots of each column. Theslides are exposed to UV light for 4 minutes to remove the cagestructures from all the ligated degenerate probes in all the sample rowsexcept rows 1 and 2, which are not illuminated.

For the third round of degenerate probe ligation, steps (a) and (b) arerepeated. Degenerate probes can only be ligated to DNA sample dots inrows 3 to 5 since the cage structure remains at the 3' end of thedegenerate probes ligated to the DNA sample dots in rows 1 and 2. Afterthese steps, the interrogation probes in rows 3 to 5 are ligated tothree degenerate probes. Then the opaque mask is laid over rows 1, 2 and3 of DNA sample dots in all of the slides (see FIG. 18D). The mask thuscovers the first, second and third sample dots of each column. Theslides are exposed to UV light for 4 minutes to remove the cagestructures from all the ligated degenerate probes in the fourth andfifth sample rows. The cage structures are not removed from thedegenerate probes in sample rows 1, 2 and 3 since they are notilluminated.

For the fourth round of degenerate probe ligation, steps (a) and (b) arerepeated. Degenerate probes can only be ligated to DNA sample dots inthe fourth and fifth rows since the cage structure remains at the 3' endof the degenerate probes ligated to the DNA sample dots in rows 1, 2 and3. After these steps, the interrogation probes in the fourth and fifthrows are ligated to four degenerate probes. Then the opaque mask is laidover rows 1, 2, 3 and 4 of DNA sample dots in all of the slides (seeFIG. 18E). The mask thus covers the first, second, third and fourthsample dots of each column. The slides are exposed to UV light for 4minutes to remove the cage structures from all the ligated degenerateprobes in the fifth sample row. The cage structures are not removed fromthe degenerate probes in sample rows 1, 2, 3 and 4 since they are notilluminated.

For the fifth round of degenerate probe ligation, steps (a) and (b) arerepeated. Degenerate probes can only be ligated to DNA sample dots inthe fifth row since the cage structure remains at the 3' end of thedegenerate probes ligated to the DNA sample dots in rows 1, 2, 3 and 4.After these steps, the interrogation probes in the fifth row are ligatedto five degenerate probes. The slides are then exposed to UV lightwithout the mask for 4 minutes to remove the cage structures from allthe ligated degenerate probes in all the sample rows. This leaves all ofthe ligated probes (which can now be considered interrogation primers)ready for chain terminating primer extension.

FIGS. 21A, 21B, 21C, 21D, 21E, 23A, 23B, 23C, 23D, and 23E depict theresults of the above degenerate probe ligation. The interrogationprimers (the top, shorter sequences following the row labels) formed byligation of degenerate probes to the interrogation probes are shownhybridized to TS-DNA (the longer sequences below each interrogationprimer) for all of the five sample dots in one column of each of thefive slides. In each slide, one additional degenerate probe has beenadded in each succeeding row, which is a consequence of successivelycovering one additional row of sample dots during each round ofdegenerate probe ligation. The non-underlined portions of theinterrogation primers represent the interrogation probe. The underlinedportions of the interrogation primers represent degenerate probesligated to the end of the interrogation probe. Careful examination ofall the interrogation primers in each set of five slides reveals thateach ends adjacent to a different nucleotide in the TS-DNA. This allowsan entire stretch of nucleotides to be separately interrogated (that is,sequenced). FIGS. 21A, 21B, 21C, 21D and 21E depict the results with anormal target sequence (that is, having 10 repeats of CA). FIGS. 23A,23B, 23C, 23D, and 23E depict the results with a mutant target sequence(that is, having only nine repeats of CA).

10. The slides are then subjected to chain terminating primer extensioncarried out for 5 minutes at 37° C. in a volume of 25 μl, under a coverslip, in the presence of 5 Units Sequenase DNA polymerase(Amersham-USB), 50 mM Tris-Cl, pH 7.5, 20 mM MgCl₂, 50 mM NaCl, 5 mMDTT, 50 μg/ml Bovine Serum Albumin (Molecular Biology grade from LifeSciences, Inc.) 50 μM fluorescent-ddATP, 50 μM fluorescent-ddCTP, 50 μMfluorescent-ddGTP, 50 μM fluorescent-ddTTP, each fluorescent dNTP beingable to generate a signal with a different emission spectrum (AppliedBiosystems, Inc. Sequencing kit). In this reaction, one fluorescentnucleotide is added to the end of each interrogation primer. Theidentity of the added nucleotide is based on the identity of thetemplate nucleotide (the nucleotide adjacent to the interrogationprimer).

11. Wash 5 minutes in 2×SSC at 25° C.

12. Wash 4 minutes in 2×SSC, 2.8% BSA, 0.12% Tween-20 at 37° C.

13. Incubate 30 minutes at 37° C. in 50 μl (under cover slip) using 5μg/ml Biotinylated AntiBUDR-Mouse.IgG (Zymed Labs) in 2×SSC, 2.8% BSA,0.12% Tween-20. This reaction collapses the TS-DNA molecules into acompact structures on the slides.

14. Wash three times for 5 minutes in 2×SSC, 2.8% BSA, 0.12% Tween-20 at37° C.

15. Incubate 30 minutes at 37° C. in 50 μl (under cover slip) usingFITC-Avidin, 5 μg/ml. in 2×SSC, 2.8% BSA, 0.12% Tween-20. This labelseach TS-DNA molecule with fluorescein.

16. Wash three time for 5 minutes in 2×SSC, 2.8% BSA, 0.12% Tween-20 at37° C.

17. Wash 5 minutes with 2×SSC, 0.01% Tween-20 at room temperature.

18. The image of each slide is captured using a microscope-CCD camerasystem with appropriate filter sets. The fluorescent emission color ofeach fluorescent nucleotide defines the nucleotide at the specificextension position in each fluorescent spot. Each spot corresponds to asingle molecule of TS-DNA. FIGS. 22A, 22B, 22C, 22D, 22E, 24A, 24B, 24C,24D, and 24E depict the results chain terminating primer extension. Theinterrogation primers, now with a fluorescent nucleotide (in boldface)added to the end, are shown hybridized to TS-DNA for all of the fivesample dots in one column of each of the five slides. In each slide, adifferent nucleotide in a stretch of nucleotides in the TS-DNA hasserved as the template for the incorporation of a chain terminatingfluorescent nucleotide. Thus, each of the nucleotides in this stretchhas been separately interrogated (that is, sequenced). FIGS. 22A, 22B,22C, 22D, and 22E depict the results with a normal target sequence (thatis, having 10 repeats of CA). FIGS. 24A, 24B, 24C, 24D, and 24E depictthe results with a mutant target sequence (that is, having only ninerepeats of CA).

19. The sequence is assembled from the fluorescent spot data obtainedfrom all five slides, by reading the incorporated nucleotide in eachrelated sample dot in the order. FIGS. 19 and 20 diagrammatically depictthe incorporated nucleotides for each sample dot in correspondingcolumns of each of the five slides. FIG. 19 represents the results withthe normal target sequence (that is, having 10 repeats of CA). FIG. 20represents the results with a mutant target sequence (that is, havingonly nine repeats of CA). The nucleotides are read first from the firstsample dot in a given column from each slide in order (that is, thesample dot in row 1 of slide 1, the sample dot in row 1 of slide 2, thesample dot in row 1 of slide 3, the sample dot in row 1 of slide 4, andthe sample dot in row 1 of slide 5). The next nucleotides are read fromthe second sample dot in the column from each slide in order (that is,the second sample dot in row 2 of slides 1 to 5 in order). The readingof nucleotides continues in the same manner for sample dots in rows 3,4, and 5 in order. The relationship of the sample dots which leads tothis order of reading can be seen by carefully examining therelationship of the interrogated nucleotides in FIGS. 22A, 22B, 22C,22D, 22E, 24A, 24B, 24C, 24D, and 24E. The sequence read from the slidesdepicted in FIG. 19 is GTGTGTGTGTGTGTGTGTGTCAATC (nucleotides 105 to 125of SEQ ID NO:25). The sequence read from the slides depicted in FIG. 20is GTGTGTGTGTGTGTGTGTCAATCTG (nucleotides 30 to 50 of SEQ ID NO:18). Thedifference in the number of GT repeats between these two sequences isreadily apparent.

Example 11 Immunoassay for Human TSH Coupled to Rolling CircleAmplification

This example describes single-molecule detection of human thyroidstimulating hormone (hTSH) using a capture antibody, and a reporterantibody. The reporter antibody is of the form illustrated in FIG. 29Awhere an antibody is coupled to a rolling circle replication primer. Thesignal that is detected is produced by rolling circle amplificationprimed by the rolling circle replication primer portion of the reporterantibody.

1. A malemide-modified monoclonal antibody specific for hTSH is coupledto the 5'-end of the 28-base oligonucleotide5'-[amino]-TTTTTTTTTTGCTGAGACATGACGAGTC-3' (SEQ ID NO:27) using SATAchemistry as described by Hendrickson et al. to form a reporterantibody. The 18 nucleotides at the 3' end of this oligonucleotide arecomplementary to the ATC described below. This oligonucleotide serves asthe rolling circle replication primer for the amplification operationbelow.

2. hTSH capture antibodies, specific for a different epitope from thatrecognized by the reporter antibody, are immobilized at definedpositions using droplets of 2 mm diameter on derivatized glass slides(Guo et al. (1994)) to make a solid-state detector. Droplets of 1.5microliters, containing 5 μg/ml of the antibody in sodium bicarbonate pH9 are applied at each defined position on the slides, incubatedovernight, and then the entire slide is washed with PBS-BLA (10 mMsodium phosphate, pH 7.4, 150 mM sodium chloride, 2% BSA, 10%Beta-lactose, 0.02% sodium azide) to block non-adsorbed sites.

3. Serial dilutions of hTSH are added to each of several identicalslides, under cover slips. After 1 hour of incubation, the slides arewashed three times with TBS/Tween wash buffer (Hendrickson et al.). ThehTSH is now captured on the surface of the glass slides.

4. Thirty microliters of appropriately diluted mixture of the reporterantibody (antibody coupled to rolling circle replication primer) isadded to each slide, under a cover slip. The slides are incubated at 37°C. for 1 hour, and then washed four times, for 5 minutes each wash, with2×SSC, 2.8% BSA, 0.12% Tween-20 at 37° C.

5. The ATC is a 94-base closed circular oligonucleotide of the followingsequence 5'-AAATCTCCAACTGGAAACTGTTCTGACTCGTCATGTCTCAGCTCTAGTACGCTGATCTCAGTCTGATCTCAGTCATTTGGTCTCAA AGTGATTG-3' (SEQ IDNO:28). This ATC oligonucleotide is incubated in a volume of 30microliters on the surface of the glass slide, under a cover slip, in abuffer consisting of 50 mM Tris-Cl, pH 7.4, 40 mM KOAC, 10 mM MgCl₂, inorder to hybridize the ATC to the rolling circle replication primerportion of the reporter antibodies. This hybridization is illustrated inFIG. 29B.

6. In situ Rolling Circle Amplification is carried out for 12 minutes at30° C., under a cover slip, in 30 microliters of a buffer containing thefollowing components: 50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 400 μM each ofdCTP, dATP, dGTP, 95 μM dTTP, 380 μM BUDR triphosphate (SIGMA), Phage T4Gene-32 protein at a concentration of 1000 nMolar, and φ29 DNApolymerase at 200 nM. This reaction generates approximately 350 tandemcopies of the ATC. The copies remain bound to the antibody as a singleTS-DNA molecule since the rolling circle replication primer isincorporated within the TS-DNA (at the 5' end) and the rolling circlereplication primer remains coupled to the antibody.

7. The slide is washed three times for 5 minutes in 2×SSC, 2.8% BSA,0.12% Tween-20 at 37° C.

8. The slide is then incubated 30 minutes at 37° C. in 50 μl (undercover slip) of 2×SSC, 2.8% BSA, 0.12% Tween-20, and 5 μg/ml BiotinylatedAntiBUDR-Mouse.IgG (Zymed Labs).

9. The slide is washed three times for 5 minutes in 2×SSC, 2.8% BSA,0.12% Tween-20 at 37° C.

10. The slide is then incubated 30 minutes at 37° C. in 50 μl (undercover slip) of 2×SSC, 2.8% BSA, 0.12% Tween-20, and FITC-Avidin at 5μg/ml.

11. The slide is washed 3×5 min. in 2×ssc, 2.8% BSA, 0.12% Tween-20 at37° C.

12. The slide is washed 10 minutes with 1×SSC, 0.01% Tween-20 at roomtemperature.

13. An image of the slide is captured using a microscope-CCD camerasystem with appropriate filter sets for fluorescein detection, and thenumber of fluorescent dots is counted. This indicates the presence of,and relative amount of, hTSH present in the sample since each dotrepresents a single collapsed TS-DNA molecule and each TS-DNA moleculerepresents a single hTSH molecule captured on the slide.

All publications cited herein, and the material for which they arecited, are hereby specifically incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 28                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 111 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GCCTGTCCAG GGATCTGCTC AAGACTCGTC ATGTCTCAGT AGCTTCTAAC GG -            #TCACAAGC     60                                                                 - - TTCTAACGGT CACAAGCTTC TAACGGTCAC ATGTCTGCTG CCCTCTGTAT T - #                111                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - GAGCAGATCC CTGGACAGGC AAGGAATACA GAGGGCAGCA GACA   - #                      - # 44                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GTATTCCTTG CCTGGTATTC CTTGCCTG         - #                  - #                 28                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - ATCAGTCTAG TCTATNNNNN            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 95 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - GAGGAGAATA AAAGTTTCTC ATAAGACTCG TCATGTCTCA GCAGCTTCTA AC -             #GGTCACTA     60                                                                 - - ATACGACTCA CTATAGGTTC TGCCTCTGGG AACAC       - #                       - #       95                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - GCTGAGACAT GACGAGTC             - #                  - #                      - #  18                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - TTTTTTTTTT TCCAACCTCC ATCACTAGT         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - TTTTTTTTTT TCCAACCTCG ATCACTAGT         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - TTTTTTTTTT TTTTTTGATC GAGGAGAAT         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - NNNNNATAGA CTAGACTGAT NNN           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 96 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - TAAAAGACTT CATCATCCAT CTCATAAGAC TCGTCATGTC TCAGCAGCTT CT -             #AACGGTCA     60                                                                 - - CTAATACGAC TCACTATAGG GGAACACTAG TGATGG      - #                       - #       96                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 96 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - TAAAAGACTT CATCATCCAT CTCATAAGAC TCGTCATGTC TCAGCAGCTT CT -            #AACGGTCA     60                                                                 - - CTAATACGAC TCACTATAGG GGAACACTAG TGATCG      - #                       - #       96                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - TTTTTTTTTT TCCAAATTCT CCTCCATCA         - #                  - #                29                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - TTTTTTTTTT TCCAAATTCT CCTCGATCA         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - TGTCCACTTT CTGTTTTCTG CCTC          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 95 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - ATCACTAGTG TTCCTTCTCA TAAGACTCGT CATGTCTCAG CAGCTTCTAA CG -             #GTCACTAA     60                                                                 - - TACGACTCAC TATAGGGGAT GATGAAGTCT TTTAT       - #                       - #       95                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - TTTTTTTTTT TTTTTTGATG GAGGAGAAT         - #                  - #                29                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - TCTCGACATC TAACGATCGA TCCTAGTGTG TGTGTGTGTG TGTCAATCTG T - #                 51                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - CTAGATACAG ATTGACACAC ACACACACAC ACACTAGGAT CGATCGTTAG AT -             #GTCGAGAT     60                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 86 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - GAACTATATT GTCTTTCTCT GTTTTCTTGC ATGGTCACAC GTCGTTCTAG TA -            #CGCTTCTA     60                                                                 - - ACTTAGTGTG ATTCCACCTT CTCNAA          - #                  - #                  86                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - AGTTTGCAGA GAAAGACAAT ATAGTTCTTK GAGAAGGTGG AATCACACTG AG - #TG               54                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - ATAGTTCTTN GAGAAGGTGG AATCACACTA AGTTAGAAG      - #                      - #    39                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 78 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - AAGTTCACGT ACGTACATAG CTAGATACAG ATTGACACAC ACACACACAC AC -             #TAGGATCG     60                                                                 - - ATCGTTAGAT GTCGAGCC             - #                  - #                      - #  78                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - AAGTTCACGT ACGTACATAG CTAGATACAG ATTGACACAC ACACACACAC AC -             #ACTAGGAT     60                                                                 - - CGATCGTTAG ATGTCGAGCC            - #                  - #                      - # 80                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 128 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - ATCTAGCTAT GTACGTACGT GAACTTTTCT TGCATGGTCA CACGTCGTTC TA -             #GTACGCTT     60                                                                 - - CTAACTTTTA ACATATCTCG ACATCTAACG ATCGATCCTA GTGTGTGTGT GT -            #GTGTGTGT    120                                                                 - - CAATCTGT                - #                  - #                       - #         128                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - CTAGATACAG ATTGACACAC ACACACACAC ACTAGGATCG ATCGTTAGAT GT -            #CGAGAT       58                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - TTTTTTTTTT GCTGAGACAT GACGAGTC         - #                  - #                 28                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 94 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - AAATCTCCAA CTGGAAACTG TTCTGACTCG TCATGTCTCA GCTCTAGTAC GC -             #TGATCTCA     60                                                                 - - GTCTGATCTC AGTCATTTGG TCTCAAAGTG ATTG       - #                  -     #        94                                                                   __________________________________________________________________________

We claim:
 1. A method of amplifying nucleic acid sequences, the methodcomprising,(a) mixing a specific binding molecule with a target samplecomprising a target molecule wherein a rolling circle replication primeris coupled to the specific binding molecule, wherein the specificbinding molecule is bound to the target molecule, (b) mixing the rollingcircle replication primer with an amplification target circle, toproduce a primer-ATC mixture, and incubating the primer-ATC mixtureunder conditions that promote hybridization between the amplificationtarget circle and the rolling circle replication primer in theprimer-ATC mixture,wherein the amplification target circle comprises asingle-stranded, circular DNA molecule comprising a primer complementportion, wherein the primer complement portion is complementary to therolling circle replication primer, and (c) mixing DNA polymerase withthe primer-ATC mixture, to produce a polymerase-ATC mixture, andincubating the polymerase-ATC mixture under conditions that promotereplication of the amplification target circle,wherein replication ofthe amplification target circle results in the formation of tandemsequence DNA.
 2. The method of claim 1 wherein the method includes atleast one of the following: (1) the use of a solid-state sample whereinthe solid-state sample comprises the target molecule, (2) a nucleic acidcollapse operation, (3) a multiplex detection operation comprisingseparately and simultaneously detecting a plurality of differentsequences present in the tandem sequence DNA, (4) differentialamplification of at least two of the amplification target circles, and(5) primer-extension sequencing.
 3. A method of amplifying nucleic acidsequences, the method comprising,(a) mixing one or more rolling circlereplication primers with one or more amplification target circles, toproduce a primer-ATC mixture, and incubating the primer-ATC mixtureunder conditions that promote hybridization between the amplificationtarget circles and the rolling circle replication primers in theprimer-ATC mixture,wherein the amplification target circles eachcomprise a single-stranded, circular DNA molecule comprising a primercomplement portion, wherein the primer complement portion iscomplementary to at least one of the rolling circle replication primers,(b) mixing DNA polymerase with the primer-ATC mixture, to produce apolymerase-ATC mixture, and incubating the polymerase-ATC mixture underconditions that promote replication of the amplification targetcircles,wherein replication of the amplification target circles resultsin the formation of tandem sequence DNA, and, simultaneous with, orfollowing, step (b), (c) mixing RNA polymerase with the polymerase-ATCmixture, and incubating the polymerase-ATC mixture under conditions thatpromote transcription of the tandem sequence DNA, wherein transcriptionof the tandem sequence DNA results in the formation of tandem sequenceRNA.
 4. A method of amplifying nucleic acid sequences, the methodcomprising,(a) mixing one or more rolling circle replication primerswith one or more amplification target circles, to produce a primer-ATCmixture, and incubating the primer-ATC mixture under conditions thatpromote hybridization between the amplification target circles and therolling circle replication primers in the primer-ATC mixture,wherein theamplification target circles each comprise a single-stranded, circularDNA molecule comprising a primer complement portion, wherein the primercomplement portion is complementary to at least one of the rollingcircle replication primers, (b) mixing DNA polymerase with theprimer-ATC mixture, to produce a polymerase-ATC mixture, and incubatingthe polymerase-ATC mixture under conditions that promote replication ofthe amplification target circles,wherein replication of theamplification target circles results in the formation of tandem sequenceDNA, (c) forming an interrogation mixture, wherein one or moreinterrogation primers are hybridized to the tandem sequence DNA, (d)simultaneous with, or following, step (c), mixing at least two differenttagged chain terminating nucleotides and DNA polymerase with theinterrogation mixture, wherein each different tagged chain terminatingnucleotide comprises a different chain terminating nucleotidetriphosphate coupled to a different tag molecule, (e) incubating theinterrogation mixture under conditions that promote template-basedaddition of the tagged chain terminating nucleotides to theinterrogation primers, wherein addition of the tagged chain terminatingnucleotides to the interrogation primers results in association of thetagged chain terminating nucleotides with the tandem sequence DNA, and(f) detecting the association of the tagged chain terminatingnucleotides with the tandem sequence DNA.
 5. The method of claim 4wherein formation of the interrogation mixture comprises(c)(1) mixing aninterrogation probe and a plurality of degenerate probes with the tandemsequence DNA to produce a probe mixture, under conditions that promotehybridization between the tandem sequence DNA and the interrogationprobe and degenerate probes, wherein each degenerate probe has a 3'blocking group, (c)(2) mixing ligase with the probe mixture, to producea degenerate ligation mixture, and incubating the degenerate ligationmixture under conditions that promote ligation of the interrogationprobe to one of the degenerate probes hybridized to the tandem sequenceDNA, wherein the degenerate probe that is ligated to the interrogationprobe is a ligated degenerate probe, (c)(3) removing the 3' blockinggroup of the ligated degenerate probe,wherein ligation of theinterrogation probe to one or more degenerate probes results in theformation of the interrogation primer, wherein the formation of theinterrogation primer results in formation of the interrogation mixture.6. The method of claim 5 wherein formation of the interrogation mixturefurther comprises, following step (c)(3),(c)(4) mixing the plurality ofdegenerate probes with the ligation mixture, to produce a secondaryprobe mixture, under conditions that promote hybridization between thetandem sequence DNA and the degenerate probes, (c)(5) mixing ligase withthe secondary probe mixture, to produce a secondary degenerate ligationmixture, and incubating the secondary degenerate ligation mixture underconditions that promote ligation of the ligated degenerate probe to oneof the degenerate probes hybridized to the tandem sequence DNA, whereinthe degenerate probe that is ligated to the ligated degenerate probe isa secondary ligated degenerate probe, (c)(6) removing the 3' blockinggroup of the secondary degenerate probe,wherein steps (c)(4), (c)(5),and (c)(6) are performed, in order, one or more times.
 7. The method ofclaim 4 wherein formation of the interrogation mixture comprises mixingan interrogation primer with the tandem sequence DNA under conditionsthat promote hybridization between the tandem sequence DNA and theinterrogation primer.
 8. A method of amplifying nucleic acid sequences,the method comprising,(a) mixing one or more rolling circle replicationprimers with one or more amplification target circles, to produce aprimer-ATC mixture, and incubating the primer-ATC mixture underconditions that promote hybridization between the amplification targetcircles and the rolling circle replication primers in the primer-ATCmixture,wherein the amplification target circles each comprise asingle-stranded, circular DNA molecule comprising a primer complementportion, wherein the primer complement portion is complementary to atleast one of the rolling circle replication primers, (b) mixing DNApolymerase with the primer-ATC mixture, to produce a polymerase-ATCmixture, and incubating the polymerase-ATC mixture under conditions thatpromote replication of the amplification target circles,whereinreplication of the amplification target circles results in the formationof tandem sequence DNA, (c) mixing a set of detection probes with thetandem sequence DNA under conditions that promote hybridization betweenthe tandem sequence DNA and the detection probes, wherein the set ofdetection probes is labeled using combinatorial multicolor coding.
 9. Amethod of amplifying nucleic acid sequences, the method comprising,(a)mixing one or more different open circle probes and one or more gapoligonucleotides with a target sample comprising one or more targetsequences, to produce an OCP-target sample mixture,wherein the targetsequences each comprise a 5' region and a 3' region, wherein the opencircle probes each comprise a single-stranded, linear DNA moleculecomprising, from 5' end to 3' end, a 5' phosphate group, a right targetprobe portion, a spacer portion, a left target probe portion, and a 3'hydroxyl group, wherein the spacer portion comprises a primer complementportion, and wherein the left target probe portion and the right targetprobe portion of the same open circle probe are each complementary tothe 3' region and the 5' region, respectively, of the same targetsequence, wherein at least one of the target sequences further comprisesa central region located between the 5' region and the 3' region,wherein neither the left target probe portion of the open circle probenor the right target probe portion of any of the open circle probes iscomplementary to the central region of the target sequences, whereineach gap oligonucleotide comprises a single-stranded, linear DNAmolecule comprising a 5' phosphate group and a 3' hydroxyl group,wherein each gap oligonucleotide is complementary all or a portion ofthe central region of at least one of the target sequences, andincubating the OCP-target sample mixture under conditions that promotehybridization between the open circle probes and the gapoligonucleotides and the target sequences in the OCP-target samplemixture, (b) mixing ligase with the OCP-target sample mixture, toproduce a ligation mixture, and incubating the ligation mixture underconditions that promote ligation of the open circle probes to formamplification target circles, (c) mixing a rolling circle replicationprimer with the ligation mixture, to produce a primer-ATC mixture, andincubating the primer-ATC mixture under conditions that promotehybridization between the amplification target circles and the rollingcircle replication primer in the primer-ATC mixture, and (d) mixing DNApolymerase with the primer-ATC mixture, to produce a polymerase-ATCmixture, and incubating the polymerase-ATC mixture under conditions thatpromote replication of the amplification target circles,whereinreplication of the amplification target circle results in the formationof tandem sequence DNA.
 10. The method of claim 9 further comprising,simultaneous with, or following, step (d),(e) mixing RNA polymerase withthe polymerase-ATC mixture, and incubating the polymerase-ATC mixtureunder conditions that promote transcription of the tandem sequence DNA,wherein transcription of the tandem sequence DNA results in theformation of tandem sequence RNA, or (e) mixing a secondary DNA stranddisplacement primer with the polymerase-ATC mixture, and incubating thepolymerase-ATC mixture under conditions that promote (i) hybridizationbetween the tandem sequence DNA and the secondary DNA stranddisplacement primer, and (ii) replication of the tandem sequence DNA inthe polymerase-ATC mixture, wherein replication of the tandem sequenceDNA results in the formation of secondary tandem sequence DNA.
 11. Themethod of claim 10 wherein the method includes at least one of thefollowing: (1) the use of a solid-state sample wherein the solid-statesample comprises the target molecule, (2) a nucleic acid collapseoperation, (3) a multiplex detection operation comprising separately andsimultaneously detecting a plurality of different sequences present inthe tandem sequence DNA, (4) differential amplification of at least twoof the amplification target circles, and (5) primer-extensionsequencing.
 12. The method of claim 9 wherein at least one of the targetsequences is coupled to a specific binding molecule, wherein thespecific binding molecule binds to a target molecule.
 13. The method ofclaim 12 wherein the method includes at least one of the following: (1)the use of a solid-state sample wherein the solid-state sample comprisesthe target molecule, (2) a step of bringing the specific bindingmolecule into contact with the target molecule, (3) a nucleic acidcollapse operation, (4) a multiplex detection operation comprisingseparately and simultaneously detecting a plurality of differentsequences present in the tandem sequence DNA, (5) differentialamplification of at least two of the amplification target circles, and(6) primer-extension sequencing.
 14. The method of claim 9 wherein atleast one of the rolling circle replication primers is coupled to aspecific binding molecule, wherein the specific binding molecule isbound to a target molecule.
 15. The method of claim 14 wherein themethod includes at least one of the following: (1) the use of asolid-state sample wherein the solid-state sample comprises the targetmolecule, (2) a step of bringing the specific binding molecule intocontact with the target molecule, (3) a nucleic acid collapse operation,(4) a multiplex detection operation comprising separately andsimultaneously detecting a plurality of different sequences present inthe tandem sequence DNA, (5) differential amplification of at least twoof the amplification target circles, and (6) primer-extensionsequencing.
 16. The method of claim 9 wherein the method furthercomprises at least one of the following: (1) a nucleic acid collapseoperation, (2) a multiplex detection operation comprising separately andsimultaneously detecting a plurality of different sequences present inthe tandem sequence DNA, (3) differential amplification of at least twoof the amplification target circles, and (4) primer-extensionsequencing.
 17. The method of claim 9 further comprisingmixing a set ofdetection probes with the tandem sequence DNA under conditions thatpromote hybridization between the tandem sequence DNA and the detectionprobes, wherein the set of detection probes is labeled usingcombinatorial multicolor coding, and detecting a plurality of differentsequences present in the tandem sequence DNA.
 18. The method of claim 9wherein the target molecule is part of a solid-state sample.
 19. Themethod of claim 18 wherein the method includes at least one of thefollowing: (1) a nucleic acid collapse operation, (2) a multiplexdetection operation comprising separately and simultaneously detecting aplurality of different sequences present in the tandem sequence DNA, (3)differential amplification of at least two of the amplification targetcircles, and (4) primer-extension sequencing.
 20. A kit for selectivelydetecting one or more target molecules, the kit comprising,(a) one ormore amplification target circles,wherein the amplification targetcircles each comprise a single-stranded, circular DNA moleculecomprising a primer complement portion, and (b) a rolling circlereplication primer comprising a single-stranded, linear nucleic acidmolecule comprising a complementary portion that is complementary to theprimer complement portion of one or more of the amplification targetcircles,wherein each amplification target circle is tethered to aspecific binding molecule, wherein the specific binding molecule bindsto at least one of the target molecules.
 21. The kit of claim 20 furthercomprising a secondary DNA strand displacement primer comprising asingle-stranded, linear nucleic acid molecule comprising a matchingportion that matches a portion of one or more of the amplificationtarget circles.
 22. The kit of claim 20 further comprising aninterrogation probe and a plurality of degenerate probes.
 23. The kit ofclaim 20 further comprising an interrogation primer.
 24. A kit forselectively amplifying nucleic acid sequences related to one or moretarget sequences, each comprising a 5' region and a 3' region, the kitcomprising,(a) one or more open circle probes each comprising asingle-stranded, linear DNA molecule comprising, from 5' end to 3' end,a 5' phosphate group, a right target probe portion, a spacer portion, aleft target probe portion, and a 3' hydroxyl group,wherein the spacerportion comprises a primer complement portion, and wherein the lefttarget probe portion is complementary to the 3' region of at least oneof the target sequences and the right target probe portion iscomplementary to the 5' region of the same target sequence, (b) arolling circle replication primer comprising a single-stranded, linearnucleic acid molecule comprising a complementary portion that iscomplementary to the primer complement portion of one or more of theopen circle probes, and (c) one or both of (1) a secondary DNA stranddisplacement primer comprising a single-stranded, linear nucleic acidmolecule comprising a matching portion that matches a portion of one ormore of the open circle probes, and (2) one or more reporter bindingagents each comprising an affinity portion and an oligonucleotideportion, wherein the oligonucleotide portion comprises one of the targetsequences.
 25. A kit for selectively amplifying nucleic acid sequencesrelated to one or more target sequences, each comprising a 5' region anda 3' region, the kit comprising,(a) one or more open circle probes eachcomprising a single-stranded, linear DNA molecule comprising, from 5'end to 3' end, a 5' phosphate group, a right target probe portion, aspacer portion, a left target probe portion, and a 3' hydroxylgroup,wherein the spacer portion comprises a primer complement portion,and wherein the left target probe portion is complementary to the 3'region of at least one of the target sequences and the right targetprobe portion is complementary to the 5' region of the same targetsequence, (b) a rolling circle replication primer comprising asingle-stranded, linear nucleic acid molecule comprising a complementaryportion that is complementary to the primer complement portion of one ormore of the open circle probes, (c) one or more gap oligonucleotides,and (d) a plurality of degenerate probes,wherein at least one of thetarget sequences further comprises a central region located between the5' region and the 3' region, wherein neither the left target probeportion of the open circle probe nor the right target probe portion ofthe open circle probe is complementary to the central region, andwherein each gap oligonucleotide comprises a single-stranded, linear DNAmolecule comprising a 5' phosphate group and a 3' hydroxyl group,wherein each gap oligonucleotide is complementary all or a portion ofthe central region of at least one of the target sequences.
 26. The kitof claim 25 further comprising an interrogation probe.
 27. The kit ofclaim 25 further comprising an interrogation primer.
 28. A method ofamplifying nucleic acid sequences, the method comprising,(a) mixing oneor more rolling circle replication primers with one or moreamplification target circles, to produce a primer-ATC mixture, andincubating the primer-ATC mixture under conditions that promotehybridization between the amplification target circles and the rollingcircle replication primers in the primer-ATC mixture,wherein theamplification target circles each comprise a single-stranded, circularDNA molecule comprising a primer complement portion, wherein the primercomplement portion is complementary to at least one of the rollingcircle replication primers, and (b) mixing DNA polymerase with theprimer-ATC mixture, to produce a polymerase-ATC mixture, and incubatingthe polymerase-ATC mixture under conditions that promote replication ofthe amplification target circles,wherein replication of theamplification target circles results in the formation of tandem sequenceDNA, wherein the tandem sequence DNA is collapsed using collapsingprobes.
 29. The method of claim 28 wherein at least one of thecollapsing probes is a collapsing detection probe.
 30. The method ofclaim 28 wherein the tandem sequence DNA is collapsed by mixing thecollapsing probes with the tandem sequence DNA, and incubating underconditions that promote hybridization between the collapsing probes andthe tandem sequence DNA.
 31. The method of claim 30 further comprising,prior to or simultaneous with the mixing of the collapsing probes withthe tandem sequence DNA, mixing detection probes with the tandemsequence DNA, and incubating under conditions that promote hybridizationbetween the detection probes and the tandem sequence DNA.
 32. The methodof claim 31 wherein the detection probes are labeled using combinatorialmulticolor coding.
 33. The method of claim 28 wherein the collapsingprobes comprise ligands, haptens, or both coupled to or incorporatedinto oligonucleotides.
 34. A method of amplifying nucleic acidsequences, the method comprising,(a) mixing one or more rolling circlereplication primers with one or more amplification target circles, toproduce a primer-ATC mixture, and incubating the primer-ATC mixtureunder conditions that promote hybridization between the amplificationtarget circles and the rolling circle replication primers in theprimer-ATC mixture,wherein the amplification target circles eachcomprise a single-stranded, circular DNA molecule comprising a primercomplement portion, wherein the primer complement portion iscomplementary to at least one of the rolling circle replication primers,and (b) mixing DNA polymerase with the primer-ATC mixture, to produce apolymerase-ATC mixture, and incubating the polymerase-ATC mixture underconditions that promote replication of the amplification targetcircles,wherein replication of the amplification target circles resultsin the formation of tandem sequence DNA, wherein the tandem sequence DNAis collapsed using ligand/ligand binding pairs, hapten/antibody pairs,or both.
 35. The method of claim 34 wherein ligands, haptens, or bothare coupled to or incorporated into the tandem sequence DNA.
 36. Themethod of claim 8 wherein at least one of the detection probes is acollapsing detection probe.
 37. The method of claim 8 furthercomprising, simultaneous with or following the mixing of the detectionprobes with the tandem sequence DNA, mixing collapsing probes with thetandem sequence DNA, and incubating under conditions that promotehybridization between the collapsing probes and the tandem sequence DNA.38. The method of claim 36 wherein the collapsing detection probecomprises ligands, haptens, or both coupled to or incorporated into anoligonucleotide.
 39. The method of claim 8 wherein the tandem sequenceDNA is collapsed using ligand/ligand binding pairs, hapten/antibodypairs, or both.
 40. The method of claim 39 wherein ligands, haptens, orboth are coupled to or incorporated into the tandem sequence DNA.
 41. Amethod of amplifying nucleic acid sequences, the method comprising,(a)mixing one or more rolling circle replication primers with one or moreamplification target circles, to produce a primer-ATC mixture, andincubating the primer-ATC mixture under conditions that promotehybridization between the amplification target circles and the rollingcircle replication primers in the primer-ATC mixture,wherein theamplification target circles each comprise a single-stranded, circularDNA molecule comprising a primer complement portion, wherein the primercomplement portion is complementary to at least one of the rollingcircle replication primers, and (b) mixing DNA polymerase with theprimer-ATC mixture, to produce a polymerase-ATC mixture, and incubatingthe polymerase-ATC mixture under conditions that promote replication ofthe amplification target circles,wherein replication of theamplification target circles results in the formation of tandem sequenceDNA, and simultaneous with, or following, step (b), (c) mixing asecondary DNA strand displacement primer with the polymerase-ATCmixture, and incubating the polymerase-ATC mixture under conditions thatpromote (i) hybridization between the tandem sequence DNA and thesecondary DNA strand displacement primer, and (ii) replication of thetandem sequence DNA in the polymerase-ATC mixture, wherein replicationof the tandem sequence DNA results in the formation of secondary tandemsequence DNA.
 42. The method of claim 41 further comprising(d) mixingRNA polymerase with the polymerase-ATC mixture, and incubating thepolymerase-ATC mixture under conditions that promote transcription ofthe secondary tandem sequence DNA, wherein transcription of thesecondary tandem sequence DNA results in the formation of tandemsequence RNA.
 43. The method of claim 42 wherein the method includes atleast one of the following: (1) a nucleic acid collapse operation, (2) amultiplex detection operation comprising separately and simultaneouslydetecting a plurality of different sequences present in the tandemsequence DNA, (3) differential amplification of at least two of theamplification target circles, and (4) primer-extension sequencing.
 44. Amethod of amplifying nucleic acid sequences, the method comprising,(a)mixing a specific binding molecule with a target sample comprising atarget molecule wherein an amplification target circle is tethered tothe specific binding molecule, wherein the specific binding moleculebinds to the target molecule, (b) mixing a rolling circle replicationprimer with the amplification target circle, to produce a primer-ATCmixture, and incubating the primer-ATC mixture under conditions thatpromote hybridization between the amplification target circle and therolling circle replication primer in the primer-ATC mixture,wherein theamplification target circle comprises a single-stranded, circular DNAmolecule comprising a primer complement portion, wherein the primercomplement portion is complementary to the rolling circle replicationprimer, and (c) mixing DNA polymerase with the primer-ATC mixture, toproduce a polymerase-ATC mixture, and incubating the polymerase-ATCmixture under conditions that promote replication of the amplificationtarget circle,wherein replication of the amplification target circleresults in the formation of tandem sequence DNA.
 45. A kit forselectively detecting one or more target molecules, the kitcomprising,(a) one or more amplification target circles,wherein theamplification target circles each comprise a single-stranded, circularDNA molecule comprising a primer complement portion, and (b) a rollingcircle replication primer comprising a single-stranded, linear nucleicacid molecule comprising a complementary portion that is complementaryto the primer complement portion of one or more of the amplificationtarget circles,wherein the rolling circle replication primer is coupledto a specific binding molecule, wherein the specific binding moleculebinds to at least one of the target molecules.
 46. The kit of claim 45further comprising a secondary DNA strand displacement primer comprisinga single-stranded, linear nucleic acid molecule comprising a matchingportion that matches a portion of one or more of the amplificationtarget circles.
 47. The method of claim 9 further comprising(e) formingan interrogation mixture, wherein one or more interrogation primers arehybridized to the tandem sequence DNA, (f) simultaneous with, orfollowing, step (e), mixing at least two different tagged chainterminating nucleotides and DNA polymerase with the interrogationmixture, wherein each different tagged chain terminating nucleotidecomprises a different chain terminating nucleotide triphosphate coupledto a different tag molecule, (g) incubating the interrogation mixtureunder conditions that promote template-based addition of the taggedchain terminating nucleotides to the interrogation primers, whereinaddition of the tagged chain terminating nucleotides to theinterrogation primers results in association of the tagged chainterminating nucleotides with the tandem sequence DNA, and (h) detectingthe association of the tagged chain terminating nucleotides with thetandem sequence DNA.
 48. The method of claim 47 wherein formation of theinterrogation mixture comprises(e)(1) mixing an interrogation probe anda plurality of degenerate probes with the tandem sequence DNA to producea probe mixture, under conditions that promote hybridization between thetandem sequence DNA and the interrogation probe and degenerate probes,wherein each degenerate probe has a 3' blocking group; (e)(2) mixingligase with the probe mixture, to produce a degenerate ligation mixture,and incubating the degenerate ligation mixture under conditions thatpromote ligation of the interrogation probe to one of the degenerateprobes hybridized to the tandem sequence DNA, wherein the degenerateprobe that is ligated to the interrogation probe is a ligated degenerateprobe; and (e)(3) removing the 3' blocking group of the ligateddegenerate probe;wherein ligation of the interrogation probe to one ormore degenerate probes results in the formation of the interrogationprimer, wherein the formation of the interrogation primer results information of the interrogation mixture.
 49. The method of claim 48wherein formation of the interrogation mixture further comprises,following step (e)(3),(e)(4) mixing the plurality of degenerate probeswith the ligation mixture, to produce a secondary probe mixture, underconditions that promote hybridization between the tandem sequence DNAand the degenerate probes; (e)(5) mixing ligase with the secondary probemixture, to produce a secondary degenerate ligation mixture, andincubating the secondary degenerate ligation mixture under conditionsthat promote ligation of the ligated degenerate probe to one of thedegenerate probes hybridized to the tandem sequence DNA, wherein thedegenerate probe that is ligated to the ligated degenerate probe is asecondary ligated degenerate probe; and (e)(6) removing the 3' blockinggroup of the secondary degenerate probe;wherein steps (e)(4), (e)(5),and (e)(6) are performed, in order, one or more times.
 50. A method ofamplifying nucleic acid sequences, the method comprising,(a) mixing oneor more different open circle probes with a target sample comprising oneor more target sequences, to produce an OCP-target samplemixture,wherein the target sequences each comprise a 5' region and a 3'region, wherein the open circle probes each comprise a single-stranded,linear DNA molecule comprising, from 5' end to 3' end, a 5' phosphategroup, a right target probe portion, a spacer portion, a left targetprobe portion, and a 3' hydroxyl group, wherein the spacer portioncomprises a primer complement portion, and wherein the left target probeportion and the right target probe portion of the same open circle probeare each complementary to the 3' region and the 5' region, respectively,of the same target sequence, wherein at least one of the targetsequences further comprises a central region located between the 5'region and the 3' region, wherein neither the left target probe portionof the open circle probe nor the right target probe portion of any ofthe open circle probes is complementary to the central region of thetarget sequences, and incubating the OCP-target sample mixture underconditions that promote hybridization between the open circle probes andthe target sequences in the OCP-target sample mixture, (b) mixing ligaseand DNA polymerase with the OCP-target sample mixture, to produce aligation mixture, and incubating the ligation mixture under conditionsthat promote ligation of the open circle probes to form amplificationtarget circles, wherein during incubation the DNA polymerase fills inthe central region of the target sequences, (c) mixing a rolling circlereplication primer with the ligation mixture, to produce a primer-ATCmixture, and incubating the primer-ATC mixture under conditions thatpromote hybridization between the amplification target circles and therolling circle replication primer in the primer-ATC mixture, and (d)mixing DNA polymerase with the primer-ATC mixture, to produce apolymerase-ATC mixture, and incubating the polymerase-ATC mixture underconditions that promote replication of the amplification targetcircles,wherein replication of the amplification target circle resultsin the formation of tandem sequence DNA.
 51. The method of claim 1further comprising, prior to step (a) binding the specific bindingmolecule to the target molecule.
 52. The method of claim 14 furthercomprising, prior to step (c) binding the specific binding molecule tothe target molecule.
 53. A method of amplifying nucleic acid sequences,the method comprising,(a) mixing one or more different open circleprobes with a target sample comprising one or more target sequences, toproduce an OCP-target sample mixture, (b) incubating the OCP-targetsample mixture under conditions that promote hybridization between theopen circle probes and the target sequences in the OCP-target samplemixture, (c) mixing ligase with the OCP-target sample mixture, toproduce a ligation mixture, and incubating the ligation mixture underconditions that promote ligation of the open circle probes to formamplification target circles, (d) mixing a rolling circle replicationprimer with the ligation mixture, to produce a primer-ATC mixture, andincubating the primer-ATC mixture under conditions that promotehybridization between the amplification target circles and the rollingcircle replication primer in the primer-ATC mixture, and (e) mixing DNApolymerase and a secondary DNA strand displacement primer with theprimer-ATC mixture, to produce a polymerase-ATC mixture, and incubatingthe polymerase-ATC mixture under conditions that promote(i) replicationof the amplification target circles, wherein replication of theamplification target circle results in the formation of tandem sequenceDNA, (ii) hybridization between the tandem sequence DNA and thesecondary DNA strand displacement primer, (iii) replication of thetandem sequence DNA, wherein replication of the tandem sequence DNAresults in the formation of secondary tandem sequence DNA, (iv)hybridization between the secondary tandem sequence DNA and the rollingcircle replication primer, and (v) replication of the secondary tandemsequence DNA, wherein replication of the tandem sequence DNA results inthe formation of tertiary tandem sequence DNA.
 54. A method ofamplifying nucleic acid sequences, the method comprising,(a) mixing oneor more rolling circle replication primers with one or moreamplification target circles, to produce a primer-ATC mixture, andincubating the primer-ATC mixture under conditions that promotehybridization between the amplification target circles and the rollingcircle replication primers in the primer-ATC mixture,wherein theamplification target circles each comprise a single-stranded, circularDNA molecule comprising a primer complement portion, wherein the primercomplement portion is complementary to at least one of the rollingcircle replication primers, and (b) mixing DNA polymerase and asecondary DNA strand displacement primer with the primer-ATC mixture, toproduce a polymerase-ATC mixture, and incubating the polymerase-ATCmixture under conditions that promote(i) replication of theamplification target circles, wherein replication of the amplificationtarget circle results in the formation of tandem sequence DNA, (ii)hybridization between the tandem sequence DNA and the secondary DNAstrand displacement primer, (iii) replication of the tandem sequenceDNA, wherein replication of the tandem sequence DNA results in theformation of secondary tandem sequence DNA, (iv) hybridization betweenthe secondary tandem sequence DNA and the rolling circle replicationprimer, and (v) replication of the secondary tandem sequence DNA,wherein replication of the tandem sequence DNA results in the formationof tertiary tandem sequence DNA.
 55. A method of amplifying nucleic acidsequences, the method comprising,(a) mixing one or more different opencircle probes with a target sample comprising one or more targetsequences, to produce an OCP-target sample mixture, (b) incubating theOCP-target sample mixture under conditions that promote hybridizationbetween the open circle probes and the target sequences in theOCP-target sample mixture, (c) mixing ligase with the OCP-target samplemixture, to produce a ligation mixture, and incubating the ligationmixture under conditions that promote ligation of the open circle probesto form amplification target circles, (d) mixing a rolling circlereplication primer with the ligation mixture, to produce a primer-ATCmixture, and incubating the primer-ATC mixture under conditions thatpromote hybridization between the amplification target circles and therolling circle replication primer in the primer-ATC mixture, and (e)mixing DNA polymerase, a secondary DNA strand displacement primer, and atertiary DNA strand displacement primer with the primer-ATC mixture, toproduce a polymerase-ATC mixture, and incubating the polymerase-ATCmixture under conditions that promote(i) replication of theamplification target circles, wherein replication of the amplificationtarget circle results in the formation of tandem sequence DNA, (ii)hybridization between the tandem sequence DNA and the secondary DNAstrand displacement primer, (iii) replication of the tandem sequenceDNA, wherein replication of the tandem sequence DNA results in theformation of secondary tandem sequence DNA, (iv) hybridization betweenthe secondary tandem sequence DNA and the tertiary DNA stranddisplacement primer, and (v) replication of the secondary tandemsequence DNA, wherein replication of the tandem sequence DNA results inthe formation of tertiary tandem sequence DNA.
 56. A method ofamplifying nucleic acid sequences, the method comprising,(a) mixing oneor more rolling circle replication primers with one or moreamplification target circles, to produce a primer-ATC mixture, andincubating the primer-ATC mixture under conditions that promotehybridization between the amplification target circles and the rollingcircle replication primers in the primer-ATC mixture,wherein theamplification target circles each comprise a single-stranded, circularDNA molecule comprising a primer complement portion, wherein the primercomplement portion is complementary to at least one of the rollingcircle replication primers, and (b) mixing DNA polymerase, a secondaryDNA strand displacement primer, and a tertiary DNA strand displacementprimer with the primer-ATC mixture, to produce a polymerase-ATC mixture,and incubating the polymerase-ATC mixture under conditions thatpromote(i) replication of the amplification target circles, whereinreplication of the amplification target circle results in the formationof tandem sequence DNA, (ii) hybridization between the tandem sequenceDNA and the secondary DNA strand displacement primer, (iii) replicationof the tandem sequence DNA, wherein replication of the tandem sequenceDNA results in the formation of secondary tandem sequence DNA, (iv)hybridization between the secondary tandem sequence DNA and the tertiaryDNA strand displacement primer, and (v) replication of the secondarytandem sequence DNA, wherein replication of the tandem sequence DNAresults in the formation of tertiary tandem sequence DNA.
 57. A methodof amplifying nucleic acid sequences, the method comprising,(a) mixingone or more different open circle probes with a target sample comprisingone or more target sequences, to produce an OCP-target sample mixture,(b) incubating the OCP-target sample mixture under conditions thatpromote hybridization between the open circle probes and the targetsequences in the OCP-target sample mixture, (c) mixing ligase with theOCP-target sample mixture, to produce a ligation mixture, and incubatingthe ligation mixture under conditions that promote ligation of the opencircle probes to form amplification target circles, (d) mixing a rollingcircle replication primer with the ligation mixture, to produce aprimer-ATC mixture, and incubating the primer-ATC mixture underconditions that promote hybridization between the amplification targetcircles and the rolling circle replication primer in the primer-ATCmixture, and (e) mixing DNA polymerase and a secondary DNA stranddisplacement primer with the primer-ATC mixture, to produce apolymerase-ATC mixture, and incubating the polymerase-ATC mixture underconditions that promote(i) replication of the amplification targetcircles, wherein replication of the amplification target circle resultsin the formation of tandem sequence DNA, (ii) hybridization between thetandem sequence DNA and the secondary DNA strand displacement primer,and (iii) replication of the tandem sequence DNA, wherein replication ofthe tandem sequence DNA results in the formation of secondary tandemsequence DNA, wherein the rolling circle replication primer is preventedfrom hybridizing to the secondary tandem sequence DNA and the secondarytandem sequence DNA is not replicated.
 58. A method of amplifyingnucleic acid sequences, the method comprising,(a) mixing one or morerolling circle replication primers with one or more amplification targetcircles, to produce a primer-ATC mixture, and incubating the primer-ATCmixture under conditions that promote hybridization between theamplification target circles and the rolling circle replication primersin the primer-ATC mixture,wherein the amplification target circles eachcomprise a single-stranded, circular DNA molecule comprising a primercomplement portion, wherein the primer complement portion iscomplementary to at least one of the rolling circle replication primers,and (b) mixing DNA polymerase and a secondary DNA strand displacementprimer with the primer-ATC mixture, to produce a polymerase-ATC mixture,and incubating the polymerase-ATC mixture under conditions thatpromote(i) replication of the amplification target circles, whereinreplication of the amplification target circle results in the formationof tandem sequence DNA, (ii) hybridization between the tandem sequenceDNA and the secondary DNA strand displacement primer, and (iii)replication of the tandem sequence DNA, wherein replication of thetandem sequence DNA results in the formation of secondary tandemsequence DNA, wherein the rolling circle replication primer is preventedfrom hybridizing to the secondary tandem sequence DNA and the secondarytandem sequence DNA is not replicated.